WO2015150465A2 - Plantes présentant une tolérance accrue aux herbicides - Google Patents

Plantes présentant une tolérance accrue aux herbicides Download PDF

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WO2015150465A2
WO2015150465A2 PCT/EP2015/057198 EP2015057198W WO2015150465A2 WO 2015150465 A2 WO2015150465 A2 WO 2015150465A2 EP 2015057198 W EP2015057198 W EP 2015057198W WO 2015150465 A2 WO2015150465 A2 WO 2015150465A2
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Prior art keywords
plant
mutated
amino acid
wildtype
polynucleotide
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PCT/EP2015/057198
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WO2015150465A3 (fr
Inventor
Raphael Aponte
Stefan Tresch
Johannes Hutzler
Thomas Mietzner
Jill Marie Paulik
Chad BROMMER
Anja Simon
Florian Vogt
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Basf Se
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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)

Definitions

  • the present invention relates in general to methods for conferring on plants agricultural level tolerance to herbicides.
  • the invention refers to plants having an increased tolerance to herbicides, more specifically to herbicides which inhibit the enzyme transketolase (TK).
  • TK transketolase
  • the present invention relates to methods and plants obtained by mutagenesis and cross-breeding and transformation that have an increased tolerance to herbicides, particularly TK-inhibiting herbicides.
  • Transketolase (EC 2.2.1.1 ) is integral to both the Calvin cycle and the oxidative pentose phosphate pathway of higher-plant chloroplasts.
  • the Calvin cycle catalyses the transfer of a two-carbon ketol group from either D-fructose- 6-phosphate or D-sedoheptulose-7- phosphate to D-glyceraldehyde- 3-phosphate to yield D-xylulose-5- phosphate and either D- erythrose-4-phosphate or D-ribose-5-phosphate, respectively [Flechner et al., Plant
  • Transketolase is thiamine pyrophosphate- dependent and is a dimer of 74 kDa subunits [de la Haba et al., J Biol Chem 214 409-426 (1955); Murphy and Walker, Planta 155, 316-320 (1982)].
  • Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined [Schenk et al., The International Journal of Biochemistry & Cell Biology 30 (1998) 1297-1318].
  • the inventors of the present invention have now surprisingly found that over-expression of wildtype and mutant transketolase forms confers in plants tolerance/resistance to particular classes of TK-inhibiting herbicides as compared to the non-transformed and/or non- mutagenized plants or plant cells, respectively. More specifically, the inventors of the present invention have found that TK expression confers tolerance/resistance to cornexistin and/or hydrocornexistin.
  • Cornexistin and hydroxycornexistin are natural products derived from the fungus
  • Paecilomyces divaricatus Isolation of cornexistin from the cultures of Paecilomyces species was published as early as 1989 by the Sankyo research group. The Sankyo Corporation discovered cornexistin during the screening of biological extracts for herbicidal use
  • hydroxycornexistin also produced in Paecilomyces variotii SANK 21086 (US00542478). Both, cornexistin and hydroxycornexistin are highly potent herbicides that have the unique quality of being harmless to corn plants. Because of this quality, both molecules have attracted research interest. Cornexistin showed good activity as a herbicide as well as relative inactivity towards corn plants. Cornexistin and hydroxycornexistin has been synthesized by chemical synthesis only as diastereomeres (Org. Biomol. Chem., 2008, 6, 4012-4025). Nine-membered carbocyclic structures in general are rare in nature and their synthesis as well as the genes involved in the synthesis is still unknown and not described.
  • the problem of the present invention can be seen as to the provision of novel traits by identifying target polypeptides, the manipulation of which makes plants tolerant to herbicides.
  • the present invention provides a plant or plant part comprising a polynucleotide encoding a wildtype or mutated TK polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to TK-inhibiting herbicides.
  • the present invention provides a seed capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention provides a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides, wherein the plant cell comprises the polynucleotide operably linked to a promoter.
  • the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in a cell, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention provides a plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the wildtype or mutated TK polypeptide conferring to the progeny or descendant plant tolerance to the TK-inhibiting herbicides.
  • the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: (a) applying an herbicide composition comprising TK-inhibiting herbicides to the locus; and (b) planting a seed at the locus, wherein the seed is capable of producing a plant that comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: applying an herbicidal composition comprising TK-inhibiting herbicides to the locus; wherein said locus is: (a) a locus that contains: a plant or a seed capable of producing said plant; or (b) a locus that is to be after said applying is made to contain the plant or the seed; wherein the plant or the seed comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • step (a) occurs before, after, or concurrently with step (b).
  • the present invention provides a method of producing a plant having tolerance to TK-inhibiting herbicides, the method comprising regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention provides a method of producing a progeny plant having tolerance to TK-inhibiting herbicides, the method comprising: crossing a first TK-inhibiting herbicides- tolerant plant with a second plant to produce a TK-inhibiting herbicides- tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides.
  • the present invention refers to a method for identifying a TK-inhibiting herbicide by using a wild-type or mutated TK of the present invention encoded by a nucleic acid which comprises the nucleotide sequence of SEQ ID NO: 182 or 183, or a variant, homologue, paralogue or orthologue thereof.
  • Said method comprises the steps of:
  • Another object refers to a method of identifying a nucleotide sequence encoding a mutated TK which is resistant or tolerant to a TK-inhibiting herbicide, the method comprising:
  • the mutated TK-encoding nucleic acid selected in step d) provides at least 2-fold as much tolerance to a TK-inhibiting herbicide as compared to that provided by the control TK-encoding nucleic acid.
  • the resistance or tolerance can be determined by generating a transgenic plant comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant.
  • Another object refers to a method of identifying a plant or algae containing a nucleic acid encoding a mutated TK which is resistant or tolerant to a TK-inhibiting herbicide, the method comprising:
  • the mutagenizing agent is ethylmethanesulfonate.
  • Another object refers to an isolated nucleic acid encoding a mutated TK, the nucleic acid comprising the sequence of SEQ ID NO: 182, or 183, or a variant thereof, as defined hereinafter.
  • the nucleic acid being identifiable by a method as defined above.
  • Another object refers to an isolated mutated TK polypeptide, the polypeptide comprising the sequence set forth in SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95
  • the present invention provides a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to TK-inhibiting herbicides, wherein the plant or plant part further exhibits a second or third herbicide-tolerant trait.
  • the invention refers to a plant cell transformed by and expressing a wild-type or a mutated TK nucleic acid according to the present invention or a plant which has been mutated to obtain a plant expressing, preferably over-expressing a wild-type or a mutated TK nucleic acid according to the present invention, wherein expression of said nucleic acid in the plant cell results in increased resistance or tolerance to a TK -inhibiting herbicide as compared to a wild type variety of the plant cell
  • the invention refers to a plant comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to TK-inhibiting herbicide as compared to a wild type variety of the plant.
  • the plants of the present invention can be transgenic or non-transgenic.
  • the expression of the nucleic acid of the invention in the plant results in the plant's increased resistance to TK-inhibiting herbicides as compared to a wild type variety of the plant
  • the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a TK-inhibiting herbicide as compared to a wild type variety of the seed.
  • the invention refers to a method of producing a transgenic plant cell with an increased resistance to a TK-inhibiting herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide.
  • the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to TK-inhibiting herbicide from the plant cell.
  • the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.
  • A, B, C, D, E, F, G, and H show a Multiple alignment using clustalw of sequences 1 to 181 depicted below in Table 1 .
  • transketolase [Solanum tuberosum] gi
  • transketolase-like protein [Arabidopsis thaliana] gi
  • an element means one or more elements.
  • control of undesired vegetation or weeds is to be understood as meaning the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired.
  • the weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds.
  • Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.
  • Monocotyledonous weeds include, but are not limited to, weeds of of the genera: Echinochloa, Setaria,
  • weeds of the present invention can include, for example, crop plants that are growing in an undesired location.
  • a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.
  • the term "plant” is used in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include but are not limited to vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes,
  • plant further encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicles, peduncles, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
  • Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
  • Averrhoa carambola e.g. Bambusa sp.
  • Benincasa hispida Bertholletia excelsea
  • Beta vulgaris Brassica spp.
  • Brassica napus e.g. Brassica napus, Brassica rapa ssp.
  • Cinnamomum spp. Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.
  • Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. e.g. Oryza sativa, Oryza latifolia
  • Syzygium spp. Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g.
  • the plant is a crop plant.
  • crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
  • the plant is a monocotyledonous plant, such as sugarcane.
  • the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
  • the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.
  • the preferred amount or concentration of the herbicide is an "effective amount” or “effective concentration.”
  • effective amount and concentration is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention.
  • the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art.
  • Herbicidal activity is exhibited by herbicides useful for the the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence.
  • the effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action.
  • a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wild-type plant.
  • herbicide-tolerant wildtype or mutated TK protein or “herbicide -resistant wildtype or mutated TK protein”
  • a TK protein displays higher TK activity, relative to the TK activity of a wild-type TK protein, when in the presence of at least one herbicide that is known to interfere with TK activity and at a
  • TK activity of such a herbicide-tolerant or herbicide- resistant mutated TK protein may be referred to herein as "herbicide-tolerant" or “herbicide- resistant” TK activity.
  • levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant.
  • the terms “herbicide-tolerant” and “herbicide-resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope.
  • the terms “herbicide-tolerance” and “herbicide-resistance” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope.
  • the terms “tolerant” and “resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope.
  • TK-inhibiting herbicides refer to those agronomically acceptable herbicide active ingredients (A.I.) recognized in the art.
  • terms such as fungicide, nematicide, pesticide, and the like refer to other agronomically acceptable active ingredients recognized in the art.
  • herbicide-tolerant and herbicide-tolerance refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of an amount of an herbicide A.I. that would normally inactivate or inhibit the activity of the wild-type (non-mutant) version of said enzyme or polypeptide.
  • TK enzyme when used specifically in regard to a TK enzyme, it refers specifically to the ability to tolerate a TK-inhibitor.
  • herbicide-tolerant wildtype or mutated TK protein or "herbicide -resistant wildtype or mutated TK protein”
  • a TK protein displays higher TK activity, relative to the TK activity of a wild-type TK protein, when in the presence of at least one herbicide that is known to interfere with TK activity and at a concentration or level of the herbicide that is known to inhibit the TK activity of the wild-type or mutated TK protein.
  • the TK activity of such a herbicide- tolerant or herbicide-resistant wildtype or mutated TK protein may be referred to herein as "herbicide-tolerant" or "herbicide-resistant” TK activity.
  • recombinant when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap- extension, or by genomic insertion or transformation.
  • a gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector.
  • the term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant.
  • transgenic plant refers to a plant that comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • a "recombinant" organism is a "transgenic” organism.
  • transgenic as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.
  • mutagenized refers to an organism or DNA thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wild-type organism or DNA, wherein the
  • human action that can be used to produce a mutagenized organism or DNA include, but are not limited to, as illustrated in regard to herbicide tolerance: tissue culture of plant cells (e.g., calli) and selection thereof with herbicides (e.g., TK-inhibiting herbicides), treatment of plant cells with a chemical mutagen such as EMS and subsequent selection with
  • GMO genetically modified organism
  • the source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).
  • mutagenized is used to refer to a plant or other organism, or the DNA thereof, in which no such transgenic material is present, but in which the native genetic material has become mutated so as to differ from a corresponding wild-type organism or DNA.
  • wild-type or “corresponding wild-type plant” means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from, e.g., mutagenized and/or recombinant forms.
  • control cell or “similar, wild-type, plant, plant tissue, plant cell or host cell” is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the herbicide-resistance characteristics and/or particular polynucleotide of the invention that are disclosed herein.
  • wild-type is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and/or does not possess herbicide-resistant
  • descendant refers to any generation plant. In some embodiments, a descendant is a first, second, third, fourth, fifth, sixth, seventh, eight, ninth, or tenth generation plant. As used herein, “progeny” refers to a first generation plant.
  • seed comprises seeds of all types, such as, for example, true seeds, caryopses, achenes, fruits, tubers, seedlings and similar forms.
  • seed refers to true seed(s) unless otherwise specified.
  • the seed can be seed of transgenic plants or plants obtained by traditional breeding methods.
  • Examples of traditional breeding methods can include cross-breeding, selfing, back- crossing, embryo rescue, in-crossing, out-crossing, inbreeding, selection, asexual propagation, and other traditional techniques as are known in the art.
  • TK-inhibiting herbicides-tolerant plant lines described as useful herein can be employed in weed control methods either directly or indirectly, i. e.
  • TK-inhibiting herbicides-tolerance trait donor lines for development, as by traditional plant breeding, to produce other varietal and/or hybrid crops containing such trait or traits.
  • All such resulting variety or hybrids crops, containing the ancestral TK-inhibiting herbicides-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, TK-inhibiting herbicides-tolerant line(s).
  • Such resulting plants can be said to retain the "herbicide tolerance characteristic(s)" of the ancestral plant, i.e. meaning that they possess and express the ancestral genetic molecular components responsible for the trait.
  • the present invention provides a plant or plant part comprising a
  • polynucleotide encoding a wildtype or mutated TK polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to TK-inhibiting herbicides.
  • the plant has been previously produced by a process comprising recombinantly preparing a plant by introducing and over-expressing a wild-type or mutated TK transgene according to the present invention, as described in greater detail hereinfter.
  • the plant has been previously produced by a process comprising in situ mutagenizing plant cells, to obtain plant cells which express a mutated TK.
  • polynucleotide encoding the wildtype or mutated TK in another embodiment, the polynucleotide encoding the wildtype or mutated TK
  • polypeptide polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 182 or 183 a variant or derivative thereof.
  • the wildtype or mutated TK polypeptide for use according to the present invention is a functional variant having, over the full-length of the variant, at least about 80%, illustratively, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 1,2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77
  • the wildtype or mutated TK polypeptide for use according to the present invention is a functional fragment of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103
  • TK polynucleotide molecules and TK polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide or an amino acid sequence that is sufficiently identical to nucleotide sequences set forth in SEQ ID Nos: 182 or 183, or to the amino acid sequences set forth in SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85
  • sequence identity refers to the extent to which two optimally aligned DNA or amino acid sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
  • identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity” is the identity fraction times 100.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG. Wisconsin Package. (Accelrys Inc. Burlington, Mass.) Polynucleotides and Oligonucleotides
  • an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state.
  • the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally
  • an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide.
  • polynucleotide(s) used interchangeably herein and refer to nucleotides, either ribonucleotides or
  • deoxyribonucleotides or a combination of both in a polymeric unbranched form of any length.
  • mutated TK nucleic acid refers to a TK nucleic acid having a sequence that is mutated from a wild-type TK nucleic acid and that confers increased TK-inhibiting herbicide tolerance to a plant in which it is expressed.
  • mutated transketolase refers to the replacement of an amino acid of the wild-type primary sequences of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99,
  • the TK nucleotide sequence encoding a mutated TK comprises the sequence of SEQ ID NO: 182, or 183, or a variant or derivative thereof Furthermore, it will be understood by the person skilled in the art that the TK nucleotide sequences encompasse homologues, paralogues and and orthologues of SEQ ID NO: 182 or 183, as defined hereinafter.
  • variants with respect to a sequence (e.g., a polypeptide or nucleic acid sequence such as - for example - a transcription regulating nucleotide sequence of the invention) is intended to mean substantially similar sequences.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein comprising the sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92
  • nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, e.g., preferably 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81 %-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide "sequence identity" to the nucleotide sequence of SEQ ID NO: 182 or 183.
  • the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides.
  • the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • substantially purified polypeptide or “purified” a polypeptide is meant that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. As the skilled addressee will appreciate, the purified polypeptide can be a recombinantly produced polypeptide.
  • polypeptide and “protein” are generally used
  • polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
  • proteins and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
  • the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids.
  • the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • the TK polypeptide of the invention comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1 %, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more
  • variant polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104,
  • Derivatives of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • functional variants and fragments of the TK polypeptides, and nucleic acid molecules encoding them also are within the scope of the present invention, and unless specifically described otherwise, irrespective of the origin of said polypeptide and irrespective of whether it occurs naturally.
  • Various assays for functionality of a TK polypeptide can be employed.
  • a functional variant or fragment of the TK polypeptide can be assayed to determine its ability to confer TK- inhibiting herbicides detoxification.
  • a TK-inhibiting herbicides detoxification rate can be defined as a catalytic rate sufficient to provide a determinable increase in tolerance to TK-inhibiting herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment of the TK polypeptide, wherein the plant or plant part expresses the variant or fragment at up to about 0.5%, illustratively, about 0.05 to about 0.5%, about 0.1 to about 0.4%, and about 0.2 to about 0.3%, of the total cellular protein relative to a similarly treated control plant that does not express the variant or fragment.
  • the wildtype or mutated TK polypeptide is a functional variant or fragment of a transketolase having the amino acid sequence set forth in SEQ ID NO: 1 or 2, wherein the functional variant or fragment has at least about 80% amino acid sequence identity to SEQ ID NO:1 or 2.
  • SEQ ID NO: 2 is identical to SEQ ID NO: 1 except that SEQ ID NO: 2 lacks the N-terminal transit peptide comprising amino acids 1-73 of SEQ ID NO: 1 [MAASSSLSTL SHHQTLLSHP KTHLPTTPAS SLLVPTTSSK VNGVLLKSTS SSRRLRVGSA SAVVRAAAVE ALE] (see Table 2a and 2b herein below for corresponding amino acid residues)
  • the functional variant or fragment further has a TK-inhibiting herbicides detoxification rate defined as a catalytic rate sufficient to provide a determinable increase in tolerance to TK-inhibiting herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.
  • a TK-inhibiting herbicides detoxification rate defined as a catalytic rate sufficient to provide a determinable increase in tolerance to TK-inhibiting herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.
  • Homologues of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded proteins without altering the biological activity of the proteins.
  • an isolated polynucleotide molecule encoding a mutated TK polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 1 or 2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues.
  • amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • a deletion refers to removal of one or more amino acids from a protein.
  • An insertion refers to one or more amino acid residues being introduced into a
  • Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S- transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag » 100 epitope, c-myc epitope, FLAG ® -epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S- transferase-tag glutathione S- transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase Tag » 100 epitope
  • c-myc epitope FL
  • a substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or ⁇ -sheet structures).
  • substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif.
  • Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).
  • Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art.
  • substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
  • “Derivatives” further include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues.
  • “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • a derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
  • reporter molecule or other ligand covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
  • derivatives also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
  • orthologues and “paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. A non-limiting list of examples of such orthologues is shown in Table 1. It will be understood by the person skilled in the art that the sequences of SEQ ID NOs:2-181 as listed in Table 1 represent orthologues and paralogues to SEQ ID NO:1.
  • paralogues and orthologues may share distinct domains harboring suitable amino acid residues at given sites, such as binding pockets for particular substrates or binding motifs for interaction with other proteins.
  • domain refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
  • motif or "consensus sequence” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage (See Figure 1 ).
  • Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
  • specific domains may also be used.
  • sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
  • Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981 ) J. Mol. Biol 147(1 );195-7).
  • 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 can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of
  • variant nucleotide sequences can be made by introducing mutations randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened to identify mutants that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
  • the inventors of the present invention have found that by substituting one or more of the key amino acid residues of the TK enzyme of SEQ ID NO: 1 or 2, e.g. by employing one of the above described methods to mutate the TK encoding nucleic acids, the tolerance or resistance to particular TK-inhibiting herbicides could be remarkably increased Preferred substitutions of mutated TK are those that increase the herbicide tolerance of the plant, but leave the biological activitiy of the oxidase activity substantially unaffected.
  • TK polypeptide comprising the sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103
  • the present sequence pattern is not limited by the exact distances between two adjacent amino acid residues of said pattern.
  • Each of the distances between two neighbours in the above patterns may, for example, vary independently of each other by up to ⁇ 10, ⁇ 5, ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions without substantially affecting the desired activity.
  • the method of site directed mutagenesis in particular saturation mutagenes (see e.g. Schenk et al., Biospektrum 03/2006, pages 277-279)
  • the inventors of the present invention have identified and generated specific amino acid subsitutions and
  • the variant or derivative of the mutated TK refers to a TK polypeptide comprising SEQ ID NO: 1 , a orthologue, paralogue, or homologue thereof, wherein the amino acid sequence differs from the wildtype amino acid sequence of a TK polypeptide at one or more positions corresponding to the following positions of SEQ ID NO:1 : 265, 267, 337, 342, 343, 458, 459, 460, 461 , 463, 51 1 , 512, 513, 514, 515, 544.
  • amino acid at or corresponding to position 265 is other than isoleucine
  • amino acid at or corresponding to position 267 is other than isoleucine
  • amino acid at or corresponding to position 337 is other than tyrosine
  • amino acid at or corresponding to position 342 is other than serine
  • amino acid at or corresponding to position 343 is other than alanine
  • amino acid at or corresponding to position 458 is other than leucine
  • amino acid at or corresponding to position 459 is other than alanine; the amino acid at or corresponding to position 460 s other than serine;
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 265 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 267 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 337 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Arg, His, Lys, Asp, Glu, Thr, Asn, Gin Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 343 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Val, Leu, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 458 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 459 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Val, Leu, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 460 is Arg, His, Lys, Asp, Glu, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 461 is Arg, His, Lys, Asp, Glu, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 51 1 is Arg, His, Lys, Asp, Glu, Ser, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Phe, Met, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 512 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 513 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 514 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Leu, lie, Phe, Met, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 515 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 544 is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, lie, Met, Phe, Tyr, or Trp.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Arg, His, Lys, Asp, Glu, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, Tyr, or Trp, and the amino acid at or
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Arg, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is His, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Lys, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Asp, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Glu, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Thr, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Asn, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Gin, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Cys, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Gly, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Pro, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Ala, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Val, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Leu, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is lie, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Met, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Phe, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Tyr, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Trp, and the amino acid at or corresponding to position 343 is Pro.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Arg, His, Lys, Asp, Glu, Thr, Asn, Gin, Cys, Gly, Pro, Ala, Val, Leu, lie, Met, Phe, Tyr, or Trp, and the amino acid at or
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Arg, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is His, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Lys, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Asp, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Glu, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Thr, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Asn, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Gin, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Cys, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Gly, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Pro, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Ala, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Val, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Leu, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is lie, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Met, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Phe, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Tyr, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 342 is Trp, and the amino acid at or corresponding to position 343 is Gly.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, Cys, Ala, Ser, Gly, and the amino acid at or corresponding to position 544 is Thr, Ala, Ser, Cys, Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, and the amino acid at or corresponding to position 544 is Ala.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Val, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 544 is Ala.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ala, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ala, and the amino acid at or corresponding to position 544 is Ala.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ala, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ala, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ala, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ser, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ser, and the amino acid at or corresponding to position 544 is Ala.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ser, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ser, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Ser, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 544 is Ala.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, Cys, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Thr, Cys, Ser, Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, Cys, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Thr, Cys, Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 51 1 is Ser, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Gly, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Thr.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Cys.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 463 is Cys, and the amino acid at or corresponding to position 511 is Ser, and the amino acid at or corresponding to position 544 is Ser.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 51 1 is Ser, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val.
  • the mutated TK polypeptide comprises a sequence of SEQ ID NO: 1 a variant, derivative, orthologue, paralogue of homologue thereof, in which: the amino acid at or corresponding to position 51 1 is Ser, and the amino acid at or corresponding to position 514 is Asn, and the amino acid at or corresponding to position 544 is Val. It will be within the knowledge of the skilled artisan to identify conserved regions and motifs shared between the homologues, orthologues and paralogues encoded by SEQ ID NO: 182 or 183, such as those depicted in Table 1.
  • amino acids corresponding to the amino acids listed below in Table 2a and 2b can be chosen to be subsituted by any other amino acid, for example by conserved amino acids, preferably by the amino acid substitutions described SUPRA using SEQ ID NO:1 as reference.
  • Table 2a and 2b provides an overview of positions in the orthologues and homologues to SEQ ID NO:1 , i.e. the corresponding positions in SEQ ID NOs: 1 to 181 .
  • Another object refers to a method of identifying a nucleotide sequence encoding a mutated TK which is resistant or tolerant to a TK-inhibiting herbicide, the method comprising:
  • the mutated TK-encoding nucleic acid selected in step d) provides at least 2-fold as much resistance or tolerance of a cell or plant to a TK-inhibiting herbicide as compared to that provided by the control TK-encoding nucleic acid.
  • the mutated TK-encoding nucleic acid selected in step d) provides at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, as much resistance or tolerance of a cell or plant to a TK-inhibiting herbicide as compared to that provided by the control TK-encoding nucleic acid.
  • the resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant or host cell, preferably a plant cell.
  • Another object refers to a method of identifying a plant or algae containing a nucleic acid comprising a nucleotide sequence encoding a wild-type or mutated TK which is resistant or tolerant to a TK-inhibiting herbicide, the method comprising:
  • said mutagenizing agent is ethylmethanesulfonate (EMS).
  • Suitable candidate nucleic acids for identifying a nucleotide sequence encoding a mutated TK from a variety of different potential source organisms including microbes, plants, fungi, algae, mixed cultures etc. as well as environmental sources of DNA such as soil. These methods include inter alia the preparation of cDNA or genomic DNA libraries, the use of suitably degenerate oligonucleotide primers, the use of probes based upon known sequences or complementation assays (for example, for growth upon tyrosine) as well as the use of mutagenesis and shuffling in order to provide recombined or shuffled mutated TK-encoding sequences.
  • Nucleic acids comprising candidate and control TK encoding sequences can be expressed in yeast, in a bacterial host strain, in an alga or in a higher plant such as tobacco or Arabidopsis and the relative levels of inherent tolerance of the TK encoding sequences screened according to a visible indicator phenotype of the transformed strain or plant in the presence of different concentrations of the selected TK-inhibiting herbicide.
  • Dose responses and relative shifts in dose responses associated with these indicator phenotypes are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed TK.
  • each mutated TK encoding sequence may be expressed, for example, as a DNA sequence under expression control of a controllable promoter such as the lacZ promoter and taking suitable account, for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different TK sequences.
  • a controllable promoter such as the lacZ promoter
  • suitable account for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different TK sequences.
  • Such strains expressing nucleic acids comprising alternative candidate TK sequences may be plated out on different concentrations of the selected TK-inhibiting herbicide in, optionally, a tyrosine supplemented medium and the relative levels of inherent tolerance of the expressed TK enzymes estimated on the basis of the extent and MIC for inhibition of the formation of the brown, ochronotic pigment.
  • candidate nucleic acids are transformed into plant material to generate a transgenic plant, regenerated into morphologically normal fertile plants which are then measured for differential tolerance to selected TK-inhibiting herbicides as described in the Example section hereinafter.
  • suitable selection markers such as kanamycin, binary vectors such as from Agrobacterium and plant regeneration as, for example, from tobacco leaf discs are well known in the art.
  • a control population of plants is likewise transformed with a nucleic acid expressing the control TK.
  • an untransformed dicot plant such as Arabidopsis or Tobacco can be used as a control since this, in any case, expresses its own endogenous TK.
  • GR50 values derived from dose/response curves having "dose” plotted on the x-axis and “percentage kill", "herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent tolerance of the expressed TK.
  • Herbicides can suitably be applied pre-emergence or post-emergence.
  • Another object of the present invention refers to an isolated nucleic acid encoding a mutated TK as disclosed SUPRA, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 182, or 183, or a variant or derivative thereof.
  • the nucleic acid is identifiable by a method as defined above.
  • the encoded mutated TK is a variant of SEQ ID NO: 1 , which includes one or more of the following:
  • amino acid corresponding to position 512 of SEQ ID NO: 1 is Arg,His,Lys,Asp,Glu,Ser,Thr,Asn,Gln,Cys,Gly,Pro,Ala,Val,Leu,lle,Met,Tyr, or Trp.
  • Trp Arg,His,Lys,Asp,Glu,Ser,Thr,Asn,Gln,Cys,Gly,Pro,Ala,Val,lle,Met,Phe,Tyr, or Trp
  • the present invention encompasses a progeny or a descendant of a TK- inhibiting herbicides-tolerant plant of the present invention as well as seeds derived from the TK-inhibiting herbicides-tolerant plants of the invention and cells derived from the TK- inhibiting herbicides-tolerant plants of the invention.
  • the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the wildtype or mutated TK polypeptide conferring to the progeny or descendant plant tolerance to the TK-inhibiting herbicides.
  • seeds of the present invention preferably comprise the TK-inhibiting herbicides-tolerance characteristics of the TK-inhibiting herbicides-tolerant plant.
  • a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the progeny or descendant plant tolerance to the TK-inhibiting herbicides.
  • plant cells of the present invention are capable of regenerating a plant or plant part. In other embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa & pericarp), and root cap.
  • the present invention provides a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to the TK-inhibiting herbicides, wherein the plant cell comprises the recombinant polynucleotide operably linked to a promoter.
  • the present invention provides a plant cell comprising a
  • polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the cell tolerance to the TK-inhibiting herbicides.
  • the invention refers to a plant cell transformed by a nucleic acid encoding a mutated TK polypeptide according to the present invention or to a plant cell which has been mutated to obtain a plant expressing a nucleic acid encoding a mutated TK polypeptide according to the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a TK-inhibiting herbicide as compared to a wild type variety of the plant cell.
  • the mutated TK polypeptide encoding nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO: 182, or 183, or a variant or derivative thereof; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78,
  • the present invention provides a plant product prepared from the TK- inhibiting herbicides-tolerant plants hereof.
  • plant products include, without limitation, grain, oil, and meal.
  • a plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed).
  • a plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the a plant or plant part tolerance to the TK-inhibiting herbicides.
  • the invention refers to a method of producing a transgenic plant cell with an increased resistance to a TK-inhibiting herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide.
  • the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a a
  • polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to TK-inhibiting herbicide from the plant cell.
  • the present invention provides a method for producing a TK-inhibiting herbicides-tolerant plant.
  • the method comprises: regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wildtype or mutated TK polypeptide encoded by the polynucleotide, the expression of the wildtype or mutated TK polypeptide conferring to the plant tolerance to the TK-inhibiting herbicides.
  • expression/expressing” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct.
  • expression in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein.
  • the process includes transcription of DNA and processing of the resulting mRNA product.
  • the at least one nucleic acid is "over-expressed” by methods and means known to the person skilled in the art.
  • increased expression or "overexpression” as used herein means any form of expression that is additional to the original wild-type expression level.
  • Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers.
  • Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest.
  • endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al.
  • nucleic acid sequences may be optimized for increased expression in a transformed plant.
  • coding sequences that comprise plant-preferred codons for improved expression in a plant can be provided. See, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1 -11 for a discussion of host-preferred codon usage. Methods also are known in the art for preparing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831 , and 5,436,391 , and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Consequently, wildtype/mutated TK nucleic acids of the invention are provided in
  • the cassette will include regulatory sequences operably linked to a mutated TK nucleic acid sequence of the invention.
  • regulatory element refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs.
  • 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
  • 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.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the wildtype/mutated TK nucleic acid sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette of the present invention will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a wildtype/mutated TK encoding nucleic acid sequence of the invention, and a transcriptional and translational 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 wildtype/mutated TK nucleic acid sequence of the invention.
  • the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “foreign” or “heterologous” to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the wildtype/mutated TK nucleic acid sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked wildtype/mutated TK nucleic acid sequence of the invention.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the native promoter sequences may be used. Such constructs would change expression levels of the wildtype/mutated TK protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked wildtype/mutated TK 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 wildtype/mutated TK nucleic acid sequence of interest, the plant host, or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens , such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991 ) Mol. Gen. Genet.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression.
  • the expression cassettes of the invention can include another selectable marker gene for the selection of transformed cells.
  • Selectable marker genes including those of the present invention, are utilized for the selection of transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin
  • HPT phosphotransferase
  • genes conferring resistance to herbicidal compounds such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D).
  • herbicidal compounds such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D).
  • sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well - characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
  • sequences can be readily modified to avoid predicted hairpin secondary mRNA structures. Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors.
  • introns of the maize Adh gene Adh1 -S intron 1 , 2, and 6 include, for example, introns of the maize Adh gene Adh1 -S intron 1 , 2, and 6 (Callis et al. Genes and Development 1 : 1 183-1200, 1987), and leader sequences, (W- sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al. Nucleic Acid Res. 15:8693-871 1 , 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990).
  • TMV Tobacco Mosaic virus
  • Maize Chlorotic Mottle Virus Maize Chlorotic Mottle Virus
  • Alfalfa Mosaic Virus Alfalfa Mosaic Virus
  • the first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs.
  • 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis.
  • the plant expression vectors of the invention also may contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.
  • MARs matrix attachment regions
  • the invention further provides an isolated recombinant expression vector comprising the expression cassette containing a wildtype/mutated TK nucleic acid nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a TK-inhibiting herbicide as compared to a wild type variety of the host cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal
  • mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., mutated TK polypeptides, fusion polypeptides, etc.)
  • Expression vectors may additionally contain 5' leader sequences in the expression construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991 ) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed.
  • TEV leader tobacco Etch Virus
  • MDMV leader Mainze Dwarf Mosaic Virus
  • BiP human immunoglobulin heavy-chain binding protein
  • AMV RNA 4 untranslated leader from the coat protein mRNA of alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382- 385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.
  • nucleic acid fragments may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with
  • tissue-preferred, or other promoters for expression in plants are constitutive, tissue-preferred, or other promoters for expression in plants.
  • Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; 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. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991 ) Theor. Appl. Genet.
  • tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue.
  • tissue-preferred promoters include, but are not limited to, leaf- preferred promoters, root-preferred promoters, seed- preferred promoters, and stem-preferred promoters.
  • tissue-preferred promoters are described by, e.g.,
  • Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3): 1331 -1341 ; Van Camp et al. (1996) Plant Physiol. 1 12(2):525-535; Canevascini et al. (1996) Plant Physiol. 1 12(2):513- 524; Yamamoto et al. (1994) Plant Cell Physiol.
  • Promoters can be modified, if necessary, for weak expression.
  • the nucleic acids of interest can be targeted to the chloroplast for expression.
  • the expression vector will additionally contain a chloroplast- targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • transit peptides are known in the art.
  • "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the desired coding sequence of the invention such that the two sequences are contiguous and in the same reading frame.
  • polypeptide of the invention by operably linking a choloroplast-targeting sequence to the 5'- end of a nucleotide sequence encoding the TK polypeptide.
  • Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991 ) J Biol. Chem. 266(5):3335-3342); EPSPS (Archer et al. (1990) J Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J Biol. Chem. 270(1 1 ):6081 -6087); plastocyanin (Lawrence et al. (1997) J Biol.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 :7301 - 7305.
  • the nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831 , herein incorporated by reference. Numerous plant transformation vectors and methods for transforming plants are available.
  • the methods of the invention involve introducing a polynucleotide construct into a plant.
  • introducing is intended presenting to the plant the polynucleotide 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 depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The term
  • introduction or “transformation” as referred to herein further means the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • stable transformation is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by
  • transient transformation is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.
  • the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell.
  • the selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.
  • the encoding nucleotide sequence is operably linked to a plant promoter, e.g. a promoter known in the art for high-level expression in a plant cell, and this construct is then introduced into a plant cell that is susceptible to TK-inhibiting herbicides; and a transformed plant is regenerated.
  • the transformed plant is tolerant to exposure to a level of TK-inhibiting herbicides that would kill or significantly injure a plant regenerated from an untransformed cell.
  • This method can be applied to any plant species or crops.
  • Methodologies for constructing plant expression vectors and introducing foreign nucleic acids into plants are generally known in the art.
  • foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors.
  • Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No.
  • nucleotide sequences into plant cells include microinjection as described by, e.g., Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by e.g., Riggs et al. (1986) Proc. Natl. Acad. ScL USA
  • Transgenic plants are preferably produced via Agrobacterium- mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1 198985 A1 , Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 -506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are
  • the nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 871 1 ).
  • Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • plants used as a model like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant)
  • crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • Agrobacterium mediated plant transformation can be performed using for example the GV3101 (pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed. - Dordrecht : Kluwer Academic Publ., 1995. - in Sect., Ringbuc
  • rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91 :694-701 ).
  • Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Patent No. 5,322,783, European Patent No. 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No.
  • Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot "The maize handbook” Springer Verlag: New York (1993) ISBN 3-540- 97826-7).
  • a specific example of maize transformation is found in U.S. Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
  • polynucleotides of the present invention may be introduced into plants by contacting plants with a virus or viral nucleic acids.
  • such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule.
  • the polypeptides of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant polypeptide.
  • promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases.
  • the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu ), saffiower (Carthamus tinctorius), wheat (Triticum aestivum, T. Turgidum ssp.
  • soybean Glycine max
  • tobacco Natural
  • Potato Potato
  • peanuts Alignment hypogaea
  • cotton Gossypium barbadense, Gossypium hirsutum
  • sweet potato Ipomoea batatus
  • cassava Manihot esculenta
  • coffee Coffea spp.
  • coconut Cocos nucifera
  • pineapple Ananas comosus
  • citrus trees Cispp.
  • Theobroma cacao tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
  • plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, saffiower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
  • crop plants for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, saffiower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.
  • transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions.
  • stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen.
  • the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the
  • flanking sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001 )
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non -transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • the expression of the nucleic acid in the plant results in the plant's increased resistance to TK-inhibiting herbicide as compared to a wild type variety of the plant.
  • the invention refers to a plant, comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to TK-inhibiting herbicide as compared to a wild type variety of the plant.
  • transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
  • a promoter for example a promoter, or
  • the natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
  • a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment.
  • transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously.
  • transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified.
  • Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place.
  • transgenic plants are mentioned herein.
  • transgenic refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering.
  • Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences.
  • the term "recombinant” does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.
  • Non-transgenic plants Plants containing mutations arising due to non-spontaneous mutagenesis and selective breeding are referred to herein as non-transgenic plants and are included in the present invention.
  • the nucleic acids can be derived from different genomes or from the same genome.
  • the nucleic acids are located on different genomes or on the same genome.
  • the present invention involves herbidicide-resistant plants that are produced by mutation breeding.
  • Such plants comprise a polynucleotide encoding a mutated TK and are tolerant to one or more TK-inhibiting herbicides.
  • Such methods can involve, for example, exposing the plants or seeds to a mutagen, particularly a chemical mutagen such as, for example, ethyl methanesulfonate (EMS) and selecting for plants that have enhanced tolerance to at least one or more TK-inhibiting herbicide.
  • EMS ethyl methanesulfonate
  • the present invention is not limited to herbicide-tolerant plants that are produced by a mutagenesis method involving the chemical mutagen EMS. Any mutagenesis method known in the art may be used to produce the herbicide-resistant plants of the present invention. Such mutagenesis methods can involve, for example, the use of any one or more of the following mutagens: radiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably from 2500 to 2900 nm), and chemical mutagens such as base analogues (e.g., 5- bromo-uracil), related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g., streptonigrin), alkylating agents (e.
  • Herbicide- resistant plants can also be produced by using tissue culture methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom. See, for example, U.S. Patent Nos. 5,773,702 and 5,859,348, both of which are herein incorporated in their entirety by reference. Further details of mutation breeding can be found in "Principals of Cultivar Development” Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference
  • the plant of the present invention comprises at least one mutated TK nucleic acid or over- expressed wild-type TK nucleic acid, and has increased tolerance to a TK-inhibiting herbicide as compared to a wild-type variety of the plant. It is possible for the plants of the present invention to have multiple wild-type or mutated TK nucleic acids from different genomes since these plants can contain more than one genome. For example, a plant contains two genomes, usually referred to as the A and B genomes. Because TK is a required metabolic enzyme, it is assumed that each genome has at least one gene coding for the TK enzyme (i.e. at least one TK gene).
  • TK gene locus refers to the position of an TK gene on a genome
  • TK gene and TK nucleic acid refer to a nucleic acid encoding the TK enzyme.
  • the TK nucleic acid on each genome differs in its nucleotide sequence from an TK nucleic acid on another genome.
  • One of skill in the art can determine the genome of origin of each TK nucleic acid through genetic crossing and/or either sequencing methods or exonuclease digestion methods known to those of skill in the art.
  • the present invention includes plants comprising one, two, three, or more mutated TK alleles, wherein the plant has increased tolerance to a TK-inhibiting herbicide as compared to a wild- type variety of the plant.
  • the mutated TK alleles can comprise a nucleotide sequence selected from the group consisting of a polynucleotide as defined in SEQ ID NO: 182 or 183, or a variant or derivative thereof, a polynucleotide encoding a polypeptide as defined in SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64
  • Allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms
  • cultivar refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered “true breeding” for a particular trait if, when the true-breeding cultivar or variety is self-pollinated, all of the progeny contain the trait.
  • breeding line or “line” refer to a group of plants within a cultivar defined by the sharing of a common set of
  • a breeding line or line is considered "true breeding" for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait.
  • the trait arises from a mutation in a TK gene of the plant or seed.
  • the herbicide-resistant plants of the invention that comprise polynucleotides encoding mutated TK polypeptides also find use in methods for increasing the herbicide-resistance of a plant through conventional plant breeding involving sexual reproduction.
  • the methods comprise crossing a first plant that is a herbicide-resistant plant of the invention to a second plant that may or may not be resistant to the same herbicide or herbicides as the first plant or may be resistant to different herbicide or herbicides than the first plant.
  • the second plant can be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant.
  • the first and second plants are of the same species.
  • the methods can optionally involve selecting for progeny plants that comprise the mutated TK polypeptides of the first plant and the herbicide resistance characteristics of the second plant.
  • the progeny plants produced by this method of the present invention have increased resistance to a herbicide when compared to either the first or second plant or both. When the first and second plants are resistant to different herbicides, the progeny plants will have the combined herbicide tolerance characteristics of the first and second plants.
  • the methods of the invention can further involve one or more generations of backcrossing the progeny plants of the first cross to a plant of the same line or genotype as either the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross can be crossed to a third plant that is of a different line or genotype than either the first or second plant.

Abstract

La présente invention se rapporte à une plante ou partie de plante comprenant un polynucléotide codant pour un polypeptide de la transcétolase de type sauvage ou mutant, l'expression dudit polynucléotide confèrant à la plante ou partie de plante une tolérance aux herbicides inhibant la transcétolase.
PCT/EP2015/057198 2014-04-03 2015-04-01 Plantes présentant une tolérance accrue aux herbicides WO2015150465A2 (fr)

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