WO2014102697A2 - Polynucléotides, polypeptides et leurs procédés d'utilisation - Google Patents

Polynucléotides, polypeptides et leurs procédés d'utilisation Download PDF

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Publication number
WO2014102697A2
WO2014102697A2 PCT/IB2013/061257 IB2013061257W WO2014102697A2 WO 2014102697 A2 WO2014102697 A2 WO 2014102697A2 IB 2013061257 W IB2013061257 W IB 2013061257W WO 2014102697 A2 WO2014102697 A2 WO 2014102697A2
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Prior art keywords
plant
cell
polynucleotide
polypeptide
sequence
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PCT/IB2013/061257
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English (en)
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WO2014102697A3 (fr
Inventor
Travis Robert Glare
John Graham Hampton
Murray Paul Cox
Mildred Marsha ORMSKIRK
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Lincoln University
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Publication of WO2014102697A2 publication Critical patent/WO2014102697A2/fr
Publication of WO2014102697A3 publication Critical patent/WO2014102697A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • the invention relates to polynucleotides, polypeptides, compositions and methods for controlling pests, particularly insect pests.
  • insect pests have been controlled by use of chemical pesticides.
  • these chemicals can be toxic to non-target species and hence can cause environmental damage.
  • Bt Bacillus thuringiensis
  • Cry2A proteins include the Cry2Adl, Cry2Aa, Cry2Ab, and Cry2Ac proteins.
  • Cry2A-like proteins and DNA sequences encoding them are also shown in U.S. Pat. No. 5,338,544, in published PCT patent application WO 00/26371 and in published PCT patent application WO 98/ 40490. Expression of Cry2A-type proteins in plants has been described in published PCT patent application WO 00/26371.
  • Bt toxins Although these Bt toxins have proven highly successful in controlling certain types of pest, they are not effective against all pest species, and have a relatively narrow target insect range compared to chemical insecticides. Therefore the search for other useful toxins and genes encoding them continues.
  • B. laterosporus an aerobic spore forming entomopathogenic bacterium, is known to be pathogenic to some insect species.
  • B. laterosporus is characterized by the formulation of a typical canoe-shaped parasporal body (CSPB) often created on one side of the spore after the sporangium lysis.
  • CSPB canoe-shaped parasporal body
  • Brembadllus laterosporus has been recorded as a pathogen of the ova and larvae of target parasitic nematodes, molluscs, and insects. Insecticidal activity of several B. lateroporus strains has been observed against the insect orders of Diptera, Coleoptera and Lepidoptera. Little is known about the virulence factors among the different strains of B. laterosporus
  • B. laterosporus against Coleoptera species was first reported in preliminary bioassays.
  • the species included the tobacco beede I ⁇ asioderma serricorne and the Colorado beetle l ⁇ ptinotarsa decemlineata (Rivers et al., 1991 , Journal of Invertebrate Pathology, 58(3), 444-447).
  • Activity against the corn rootworm, Oiabratica spp. has been reported for different strains of B.
  • Lepidopteran activity against the velvetbean caterpillar Anticarsia gemmatalis was reported in the same study. It would be beneficial to have multiple toxins to target different insect species, or even the same target species because the use of toxins having different modes of action may help prevent or delay the development of insect resistance. It would also be beneficial to have toxins that are active against different ranges of insect groups for use with different crops depending on the target species present. It is an object of the invention to provide at least one novel toxin protein and/ or at least to provide the public with a useful choice.
  • the invention provides an isolated polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO: 1 to 58 or a variant or fragment thereof.
  • the variant has at least 70% identity to the sequence of any one of SEQ ID NO: 1 to 58.
  • the fragment comprises at least 20 contiguous amino acids of the sequence of any one of SEQ ID NO: 1 to 58.
  • polypeptide, variant or fragment has insecticidal activity.
  • polypeptide, variant or fragment has insecticidal activity against at least one of:
  • the polypeptide, variant or fragment has insecticidal activity against at least one Lepidoptera species, and at least one Diptera species.
  • the at least one Lepidoptera species is diamondback moth ⁇ Plutella xylostelld).
  • the at least one Lepidoptera species is cabbage looper moth (Tnchoplusia ni).
  • the at least one Lepidoptera is a Torticidae or a Plutellidae.
  • the at least one Diptera species is a mosquito species.
  • mosquito species is selected from Culex pervigilans and Opifex fuscus.
  • polypeptide, variant or fragment is active against both
  • diamondback moth Plutella xylostelld
  • mosquito species selected from Culex pervi ilans and Opifex fuscus.
  • the polynucleotide comprises a sequence selected from any one of SEQ ID NO: 59 to 116 or a fragment or variant thereof.
  • the variant comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 59 to 116.
  • the fragment comprises at least 60 contiguous nucleotides of the sequence of any one of SEQ ID NO: 59 to 116.
  • an isolated polynucleotide comprises a sequence selected from any one of SEQ ID NO: 59 to 116, or a variant or fragment thereof.
  • the variant comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 59 to 116.
  • the fragment comprises at least 60 contiguous nucleotides of the sequence of any one of SEQ ID NO: 59 to 116.
  • the invention provides a genetic construct comprising at least one polynucleotide of the invention.
  • polynucleotide of the invention is operably linked to a promoter polynucleotide. In one embodiment the polynucleotide of the invention and the promoter polynucleotide are not normally associated in nature.
  • Cells in a further embodiment the invention provides a cell comprising at least one polynucleotide of the invention.
  • the invention provides a cell comprising at least one genetic construct of the invention.
  • the cell is transgenic for the at least one polynucleotide of the invention, or the at least one genetic construct of the invention.
  • the cell, or it precursor cell is transformed with the at least one
  • polynucleotide or the at least one construct of the invention.
  • the cell expresses the polypeptide.
  • polypeptide when expressed in the cell has insecticidal activity.
  • polypeptide when expressed in the cell has insecticidal activity against at least one of:
  • the cell when expressing the polypeptide has insecticidal activity against at least one of:
  • the cell has increased insecticidal activity relative to a control cell.
  • the control cell may be any cell of the same type that is not transformed with the
  • polynucleotide, or construct, of the invention to express the polypeptide.
  • control cell is an untransformed cell.
  • Plant in a further embodiment the invention provides a plant cell or plant comprising at least one polynucleotide of the invention.
  • the invention provides a plant cell or plant comprising at least one genetic construct of the invention.
  • the plant cell or plant expresses at least one polypeptide of the invention.
  • polypeptide when expressed in the plant cell or plant has insecticidal activity.
  • polypeptide when expressed in the plant cell or plant has insecticidal activity against at least one of:
  • the plant cell or plant, when expressing the polypeptide has insecticidal activity. In one embodiment the plant cell or plant, when expressing the polypeptide has insecticidal activity against at least one of:
  • polynucleotide of the invention is operably linked to a promoter polynucleotide that drives expression preferentially in a particular tissue, organ, cell or organelle.
  • polypeptide when expressed in the particular tissue, organ, cell or organelle has insecticidal activity.
  • polypeptide when expressed in the particular tissue, organ, cell or organelle, has insecticidal activity against at least one of:
  • the particular tissue, organ, cell or organelle, when expressing the polypeptide has insecticidal activity. In one embodiment the particular tissue, organ, cell or organelle, when expressing the polypeptide has insecticidal activity against at least one of:
  • the cell or plant of the invention comprises, is transgenic for, or is transformed with at least two polynucleotides of the invention.
  • the two polynucleotides are a first polynucleotide and a second polynucleotide.
  • the first polynucleotide is selected from the sequence of any one of SEQ ID NO: 62 and 63
  • the second polynucleotide is selected from the sequence of any one of SEQ ID NO: 64 and 65.
  • the first polynucleotide is selected from the sequence of any one of SEQ ID NO: 67 and 68
  • the second polynucleotide is selected from the sequence of any one of SEQ ID NO: 69 to 72.
  • the cell or plant of the invention comprises, or expresses at least two polypeptides of the invention.
  • the two polypeptides are a first polypeptide and a second polypeptide.
  • first polypeptide is selected from the sequence of any one of SEQ ID NO: 4 and 5
  • second polypeptide is selected from the sequence of any one of SEQ ID NO: 6 and 7.
  • first polypeptide is selected from the sequence of any one of SEQ ID NO: 9 and 10
  • second polypeptide is selected from the sequence of any one of SEQ ID NO: 11 to 14.
  • first and second polypeptides are binary toxin partners. In a further embodiment both polypeptides forming the binary toxin pair are required for insecticidal activity.
  • the invention provides a polypeptide encoded by a polynucleotide of the invention.
  • the invention provides an isolated polypeptide comprising the sequence of any one of SEQ ID NO: 1 to 58 or a variant or fragment thereof.
  • the variant has at least 70% identity to the sequence of any one of SEQ ID NO: 1 to 58.
  • the fragment comprises at least 20 contiguous amino acids of the sequence of any one of SEQ ID NO: 1 to 58.
  • the polypeptide, variant or fragment has insecticidal activity
  • polypeptide, variant or fragment has insecticidal activity against at least one of:
  • polypeptide, variant or fragment has insecticidal activity against at least one Lepidoptera species, and at least one Diptera species.
  • the at least one Lepidoptera species is diamondback moth ⁇ Plutella xylostelld). In one embodiment the at least one Lepidoptera species is cabbage looper moth (Tnchoplusia ni). In one embodiment the at least one Lepidoptera is a Torticidae or a Plutellidae. In a further embodiment the at least one Diptera species is a mosquito species. In a further embodiment the mosquito species is selected from Culex pervigilans and Opifex fuscus.
  • polypeptide, variant or fragment is active against both
  • the invention provides a polynucleotide encoding a polypeptide of the invention.
  • the invention provides a method for making an insect resistant plant cell or plant, the method comprising the step of transforming a plant cell or plant with at least one polynucleotide of the invention.
  • the polynucleotide is expressed to produce an encoded polypeptide of the invention which has insecticidal activity and confers insect resistance on the plant cell or plant.
  • polypeptide when expressed in the plant cell or plant has insecticidal activity.
  • polypeptide when expressed in the plant cell or plant has insecticidal activity against at least one of:
  • the plant when expressing the polypeptide has insecticidal activity.
  • the plant cell or plant when expressing the polypeptide has insecticidal activity against at least one of:
  • polynucleotide of the invention is operably linked to a promoter polynucleotide that drives expression preferentially in a particular tissue, organ, cell or organelle.
  • polypeptide when expressed in the particular tissue, organ, cell or organelle has insecticidal activity.
  • polypeptide when expressed in the particular tissue, organ, cell or organelle, has insecticidal activity against at least one of:
  • the particular tissue, organ, cell or organelle, when expressing the polypeptide has insecticidal activity.
  • the particular tissue, organ, cell or organelle when expressing the polypeptide has insecticidal activity against at least one of: iii) at least one Lepidoptera species, and
  • the plant cell or plant is transformed with at least two polynucleotides of the invention.
  • the two polynucleotides are a first polynucleotide and a second polynucleotide.
  • the first polynucleotide is selected from the sequence of any one of SEQ ID NO: 62 and 63
  • the second polynucleotide is selected from the sequence of any one of SEQ ID NO: 64 and 65.
  • the first polynucleotide is selected from the sequence of any one of SEQ ID NO: 67 and 68
  • the second polynucleotide is selected from the sequence of any one of SEQ ID NO: 69 to 72.
  • the plant produced by the method expresses at least two polypeptides of the invention.
  • the two polypeptides are a first polypeptide and a second polypeptide.
  • the first polypeptide is selected from the sequence of any one of SEQ ID NO: 4 and 5
  • the second polypeptide is selected from the sequence of any one of SEQ ID NO: 6 and 7.
  • first polypeptide is selected from the sequence of any one of SEQ ID NO: 9 and 10
  • second polypeptide is selected from the sequence of any one of SEQ ID NO: 11 to 14.
  • first and second polypeptides are binary toxin partners. In a further embodiment both polypeptides forming the binary toxin pair are required for insecticidal activity.
  • the invention provides a plant cell or plant produced by a method of the invention.
  • the invention provides a composition comprising at least one polypeptide, variant or fragment of the invention.
  • the composition comprises an agriculturally acceptable carrier.
  • composition has insecticidal activity.
  • Lepidoptera species and at least one Diptera species.
  • the at least one Lepidoptera species is diamondback moth ⁇ Plutella xylostelld). In one embodiment the at least one Lepidoptera species is cabbage looper moth (Tnchoplusia nt). In one embodiment the at least one Lepidoptera is a Torticidae or a Plutellidae. In a further embodiment the at least one Diptera species is a mosquito species. In a further embodiment the mosquito species is selected from Culex pervigilans and Opifex fuscus.
  • composition is active against both diamondback moth (Plutella xylostelld) and at least one mosquito species selected from Culex pervigilans and Opifex fuscus.
  • diamondback moth Plutella xylostelld
  • mosquito species selected from Culex pervigilans and Opifex fuscus.
  • composition comprises at least two polypeptides of the invention.
  • two polypeptides are a first polypeptide and a second polypeptide.
  • first polypeptide is selected from the sequence of any one of SEQ ID NO: 4 and 5
  • second polypeptide is selected from the sequence of any one of SEQ ID NO: 6 and 7.
  • first polypeptide is selected from the sequence of any one of SEQ ID NO: 9 and 10
  • second polypeptide is selected from the sequence of any one of SEQ ID NO: 11 to 14.
  • first and second polypeptides are binary toxin partners. In a further embodiment both polypeptides forming the binary toxin pair are required for insecticidal activity.
  • the invention provides a part, propagule or progeny of a plant of the invention.
  • the part, propagule or progeny comprises at least one of a polynucleotide, construct, polypeptide, variant or fragment of the invention.
  • the part, propagule or progeny expresses a polypeptide, variant or fragment of the invention.
  • the part is from a vegetative tissue. In one embodiment the part is a leaf. In a further embodiment the part is a root.
  • the invention provides meal prepared from at least one of a cell, plant cell, plant part, propagule and progeny of the invention.
  • the invention provides meal comprising at least one of a cell, plant cell, plant part, propagule and progeny of the invention.
  • Animal feed In a further aspect the invention provides an animal feedstock prepared from at least one of a polynucleotide, construct, cell, plant cell, plant part, propagule and progeny of the invention.
  • the invention provides an animal feedstock comprising at least one of a cell, plant cell, plant part, propagule and progeny of the invention
  • the invention provides a biofuel feedstock prepared from at least one of a polynucleotide, construct, cell, plant cell, plant part, propagule and progeny of the invention.
  • the invention provides a biofuel feedstock comprising at least one of a polynucleotide, construct, cell, plant cell, plant part, propagule and progeny of the invention.
  • Insect control method
  • the invention provides a method of controlling at least one insect insect the method comprising expressing at least one polypeptide of the invention in at least one plant cell or plant.
  • At least one insect feeding on the at least plant cell or plant is controlled by the at least one polypeptide.
  • At least one insect is killed by ingesting the at least one polypeptide.
  • insects covers all liquid and solid carriers known in the art such as water and oils, as well as adjuvants, dispersants, binders, wettants, surfactants, humectants tackifiers, and the like that are ordinarily known for use in the preparation of control compositions, including insecticide compositions.
  • insecticidal activity means activity in at least one of: killing, slowing the growth of, preventing reproduction of, and reducing numbers of any given insect.
  • insect resistant plant means that the plant is resistant to the presence of insect pests than a control plant.
  • the phrase "insect resistant plant” means that the plant has less insect damage in the presence of an insect pest than a control plant in the presence of the same insect pest. In a preferred embodiment the phrase “insect resistant plant” means that the plant has at least one of increased yield, increased growth rate, increased biomass, increased seed yield, in the presence of an insect pest than a control plant in the presence of the same insect pest.
  • a control plant is a plant of the same type that is not expressing the polypeptide of the invention that is expressed in the insect resistant plant.
  • An "insect pest” is an insect that causes damage to a non-insect resistant plant.
  • the cell is a prokaryotic cell. In a further embodiment the cell is a eukaryotic cell. In one embodiment the cell is selected from a bacterial cell, a yeast cell, a fungal cell, an insect cell, algal cell, and a plant cell. In one embodiment the cell is a bacterial cell. In a further embodiment the cell is a yeast cell. In one embodiment the yeast cell is a S. ceriviseae cell. In further embodiment the cell is a fungal cell. In further embodiment the cell is an insect cell. In further embodiment the cell is an algal cell. In a further embodiment the cell is a plant cell.
  • the cell is a non-plant cell.
  • the non-plant is selected from E. coli, P. pastoris, S. ceriviseae, D. salina, C. reinhardtii.
  • the non-plant is selected from P. pastoris, S. ceriviseae, D. salina, C. reinhardtii.
  • the cell is a microbial cell.
  • the microbial cell is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates).
  • the microbial cell is an algal cell of the species Chlamydomo nas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridium, Phaeodacyt lum,
  • the algal cell is Chlamydomo nas reinhardtii.
  • the cell is from the genus Yarroma, Candida, hodotorula, hodosporidium, Cryptococcus, Trichosporon, Upomyces, Pythium, Thraustochytrium, or Ulkenia.
  • the cell is a bacterium of the genus hodococcus, Escherichia, or a cyanobacterium.
  • the cell is a yeast cell.
  • the cell is a synthetic cell.
  • the plant cells, and plants in which the polynucleodes and polypeptides of the invention are expressed may be from any plant species.
  • the plant cell or plant is from a gymnosperm plant species.
  • the plant cell or plant is from an angiosperm plant species. In a further embodiment the plant cell or plant, is from a from dicotyledonous plant species.
  • Preferred dicotyledonous genera include: Amygdalus, Anacardium, Anemone, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Claytonia, Coriandrum, Coronilla, Corydalis, Crotalaria, Cyclamen, Dentaria, Dicentra, Dolichos, Eranthis, Glycine, Gossypium, Helianthus, Lathyrus, Lens, Lespede ⁇ a, Unum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Oxalis, Papaver, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Kibes, Ricinus, Sesamum, Thalictrum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.
  • Preferred dicotyledonous species include: Amygdalus communis, Anacardium occidentale, Anemone americana, Anemone occidentalis, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica nigra, Brassica campestris, Cajanus cajan, Cajanus indicus, Cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Claytonia exigua, Claytonia megarhi ⁇ a, Coriandrum sativum, Coronilla ⁇ , Corydalis flavula, Corydalis sempervirens, Crotalaria juncea, Cyclamen coum, Dentaria laciniata, Dicentra eximia, Dicentra formosa, Dolichos lablab, Eranthis hyemalis, Gossypium arboreum, Gossypium nank
  • the plant cell or plant is from a monocotyledonous plant species.
  • Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Aparagus, Avena, Bambusa, Bellavalia, Brimeura, Brodiaea, Bulbocodium, Bothrichloa, Bouteloua, Bromus, Calamovilfa, Camassia, Cenchrus, Chionodoxa, Chloris, Colchicum, Crocus, Cymbopogon, Cynodon, Cypripedium, Dactylis, Dichanthium, Digitaria, Elaeis, Eleusine, Eragrostis, Eremurus, Erythronium, Fagopyrum, Festuca, Fritillaria, Galanthus, Helianthus, Hordeum, Hyacinthus, Hyacinthoides, Ipheion, Iris, Feucojum, Fiatris,
  • Preferred monocotyledonous species include: Agropyron cristatum, Agropyron desertorum, Agropyron elongatum, Agropyron intermedium, Agropyron smithii, Agropyron spicatum, Agropyron trachycaulum, Agropyron trichophorum, Allium ascalonicum, Allium cepa, Allium chinense, Allium porrum, Allium schoenoprasum, Allium fistulosum, Allium sativum, Alopecurus pratensis, Andropogon gerardi, Andropogon Gerardii, Andropogon scoparious, Arrhenatherum elatius, Aparagus officinalis, Avena nuda, Avena sativa, Bambusa vulgaris, Bellevalia trifoliate, Brimeura amethystina, Brodiaea californica, Brodiaea coronaria, Brodiaea elegans
  • Preferred plants include crop plants, such as cotton, sorghum, maize, wheat, rice, soy and barley.
  • leguminous plants are leguminous plants.
  • the leguminous plant or part thereof may encompass any plant in the plant family Leguminosae or Fabaceae.
  • the plants may be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including, beans, lentils, lupins, peas, peanuts, soy bean; bloom legumes including lupin, pharmaceutical or industrial legumes; and fallow or green manure legume species.
  • Ar preferred genus is Glycine.
  • Preferred Glycine species include Glycine max and Glycine wightii (also known as Neonotonia wightii).
  • a particularly preferred Glycine species is Glycine max, commonly known as soy bean.
  • a particularly preferred Glycine species is Glycine wightii, commonly known as perennial soybean.
  • Visum Another preferred genus is Visum.
  • a preferred Visum species is Visum sativum commonly known as pea.
  • Another preferred genus is Brassica.
  • a preferred Brassica species is Brassica oleracea, commonly known as forage kale and cabbage.
  • Another preferred genus is Sorghum.
  • Preferred Sorghum species include Sorghum bicolor, Sorghum bathna, Sorghum halepense, and Sorghum sudanense.
  • a preferred species is Sorghum bicolor. Another preferred genus is 7 * ea. A preferred 7.ea species is 7 * ea mays.
  • a preferred grain producing genera is Hordeum.
  • a preferred grain producing species is Hordeum vulgare.
  • a preferred biofuel genera is Miscanthus.
  • a preferred biofuel species is Miscanthus giganteus.
  • ferred biofuel genera is Saccharum.
  • a preferred biofuel species is Saccharum ofricinarum.
  • a preferred biofuel genera is Panicum.
  • a preferred biofuel species is Panicum mrgatum.
  • plant is intended to include a whole plant, any part of a plant, a seed, a fruit, propagules and progeny of a plant.
  • 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
  • plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting progeny, comprising the polynucleotides or constructs of the invention, and/ or expressing the polypeptide sequences of the invention, also form an part of the present invention.
  • the plants, plant parts, propagules and progeny comprise a polynucleotide or construct of the invention, and/ or express a polypeptide sequence of the invention.
  • polynucleotide(s), means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.
  • a “fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides.
  • the term “primer” refers to a short polynucleotide, usually having a free 3'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.
  • probe refers to a short polynucleotide that is used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay.
  • the probe may consist of a "fragment" of a polynucleotide as defined herein.
  • polypeptide encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.
  • Polypeptides of the present invention, or used in the methods of the invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide that preferably performs a function of and/ or provides three dimensional structure of the polypeptide.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.
  • isolated as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment.
  • An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
  • recombinant refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/ or is recombined with sequences that are not present in its natural context.
  • a "recombinant" polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.
  • polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species.
  • the polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinandy.
  • variant refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variant with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.
  • Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57% more preferably at least 58%, more preferably at least 59%, more preferably at least 60% more preferably at least 61%, more preferably at least 62%, more preferably at least 63% more preferably at least 64%, more preferably at least 65%, more preferably at least 66% more preferably at least 67%, more preferably at least 68%, more preferably at least 69% more preferably at least 70%, more preferably at least 71%, more preferably at least 72% more preferably at least 73%, more preferably at least 74%, more preferably at least 75% more preferably at least 76%, more preferably at least 77%, more preferably at least 78% more preferably at least 79%, more preferably at least 80%, more preferably at least 8
  • Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.
  • Polynucleotide sequence identity can be determined in the following manner.
  • the subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from the NCBI website on the World Wide Web at ftp:/ /ftp.ncbi.nih.gov/blast/.
  • the default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
  • polynucleotide sequences may be examined using the following unix command line parameters: bl2seq— i nucleotideseql— j nucleotideseq2— F F— p blastn
  • the parameter— F F turns off filtering of low complexity sections.
  • the parameter— p selects the appropriate algorithm for the pair of sequences.
  • Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453).
  • a full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from the world wide web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/.
  • the European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www. ebi.ac.uk/ emboss/ align/.
  • GAP Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • a preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)
  • Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/.
  • the parameter— F F turns off filtering of low complexity sections.
  • the parameter— p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
  • Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably less than 1 x 10-100 when compared with any one of the specifically identified sequences.
  • variant polynucleotides of the present invention, or used in the methods of the invention hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.
  • hybridize under stringent conditions refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • a target polynucleotide molecule such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot
  • the ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
  • Tm melting temperature
  • Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500 /oligonucleotide length) 0 C.
  • PNAs peptide nucleic acids
  • Tm values are higher than those for DNA-DNA or DNA- RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6.
  • Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.
  • Variant polynucleotides of the present invention, or used in the methods of the invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention.
  • a sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
  • Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significandy altering its biological activity are also included in the invention.
  • a skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
  • Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp:/ / ftp.ncbi.nih.gov/blast/ via the tblastx algorithm as previously described.
  • Polypeptide variants may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp:/ / ftp.ncbi.nih.gov/blast/ via the tblastx algorithm as previously described.
  • variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more
  • Polypeptide sequence identity can be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from the NCBI website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/.
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from the NCBI website on the World Wide Web at ftp://ftp.ncbi.nih.gov/blast/.
  • the default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
  • Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs.
  • EMBOSS-needle available at http:/www.ebi.ac.uk/emboss/align/
  • GAP Human, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
  • a preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)
  • Polypeptide variants of the present invention, or used in the methods of the invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the
  • Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably 1x10- 100 when compared with any one of the specifically identified sequences.
  • the parameter— F F turns off filtering of low complexity sections.
  • the parameter— p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
  • the term "genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.
  • a genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/ or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA.
  • the genetic construct may be linked to a vector.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell.
  • the vector may be capable of replication in at least one additional host system, such as E. colt.
  • expression construct refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
  • An expression construct typically comprises in a 5' to 3' direction:
  • coding region or "open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/ or a polypeptide under the control of appropriate regulatory sequences.
  • the coding sequence may, in some cases, identified by the presence of a 5' translation start codon and a 3' translation stop codon.
  • a "coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • “Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
  • noncoding region refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination, mRNA stability, and for regulation of translation efficiency.
  • Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
  • promoter refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors. Introns within coding sequences can also regulate transcription and influence post-transcriptional processing (including splicing, capping and polyadenylation).
  • a promoter may be homologous with respect to the polynucleotide to be expressed. This means that the promoter and polynucleotide are found operably linked in nature.
  • the promoter may be heterologous with respect to the polynucleotide to be expressed. This means that the promoter and the polynucleotide are not found operably linked in nature.
  • polynucleotides/polypeptides of the invention may be andvantageously expessed under the control of selected promoter sequences as described below.
  • Vegetative tissue spedfic promoters An example of a vegetative specific promoter is found in US 6,229,067; and US 7,629,454; and US 7,153,953; and US 6,228,643.
  • Pollen spedfic promoters An example of a pollen specific promoter is found in US 7,141,424; and US 5,545,546; and US 5,412,085; and US 5,086,169; and US 7,667,097.
  • a seed specific promoter is found in US 6,342,657; and US 7,081,565; and US 7,405,345; and US 7,642,346; and US 7,371,928.
  • a preferred seed specific promoter is the napin promoter of Brassica napus (Josefsson et al., 1987, J Biol Chem. 262(25):12196-201; EUerstrom et al., 1996, Plant Molecular Biology, Volume 32, Issue 6, pp 1019-1027).
  • Non-photosynthetic tissue preferred promoters include those preferentially expressed in nonphotosynthetic tissues / organs of the plant.
  • Non-photosynthetic tissue preferred promoters may also include light repressed promoters.
  • Photosythetic tissue preferred promoters include those that are preferentially expressed in photosynthetic tissues of the plants.
  • Photosynthetic tissues of the plant include leaves, stems, shoots and above ground parts of the plant.
  • Photosythetic tissue preferred promoters include light regulated promoters.
  • Light regulated promoters are known to those skilled in the art and include for example chlorophyll a/b (Cab) binding protein promoters and Rubisco Small Subunit (SSU) promoters.
  • An example of a light regulated promoter is found in US 5,750,385.
  • Light regulated in this context means light inducible or light induced.
  • transgene is a polynucleotide that is taken from one organism and introduced into a different organism by transformation.
  • the transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
  • Host cells may be derived from, for example, bacterial, fungal, yeast, insect, mammalian, algal or plant organisms. Host cells may also be synthetic cells. Preferred host cells are eukaryotic cells. A particularly preferred host cell is a plant cell, particularly a plant cell in a vegetative tissue of a plant. A "transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
  • polypeptides of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.
  • PCR polymerase chain reaction
  • the polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
  • hybridization probes include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes.
  • Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of twenty minutes each at 55°C) in 1.
  • An optional further wash (for twenty minutes) can be conducted under conditions of 0.1 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C.
  • polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR
  • a partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based method, computer/ database—based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene.
  • the fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Divergent primers are designed from the known region.
  • standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
  • Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
  • the polynucleotide sequence of a primer useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
  • Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies. Computer based methods
  • variant sequences of the invention may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
  • An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp:/ /ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases.
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
  • BLASTP compares an amino acid query sequence against a protein sequence database.
  • BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database.
  • tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
  • tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • BLAST family of algorithms including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • the BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see by chance when searching a database of the same size containing random contiguous sequences.
  • the Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • CLUSTALW Thimpson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, http:/ /www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE (Cedric
  • Pattern recognition software applications are available for finding motifs or signature sequences.
  • MEME Multiple Em for Motif Elicitation
  • MAST Motif Alignment and Search Tool
  • the MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found.
  • MEME and MAST were developed at the University of California, San Diego.
  • PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences.
  • the PROSITE database www.expasy.org/prosite
  • Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
  • polypeptides of the invention may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, California).
  • peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, California).
  • Mutated forms of the polypeptides may also be produced during such syntheses.
  • polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification).
  • polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/ or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined.
  • the invention provides a host cell which comprises a genetic construct or vector of the invention.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention.
  • Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
  • the expressed recombinant polypeptide which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
  • the invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention, or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.
  • strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant ceU, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a ceU, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed.
  • the expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
  • Genetic constructs for expression of genes in transgenic plants typicaUy include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.
  • the promoters suitable for use in the constructs of this invention are functional in a ceU, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, ceU cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired.
  • the promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi.
  • promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention.
  • constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize.
  • Plant promoters which are active in specific tissues respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894 and WO2011 / 053169, which is herein incorporated by reference.
  • Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobactenum tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Ory ⁇ a sativa ADP-glucose pyrophosphorylase terminator and the Solatium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobactenum tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zein gene terminator
  • the Ory ⁇ a sativa ADP-glucose pyrophosphorylase terminator the Solatium tuberosum PI-II terminator.
  • NPT II neomycin phophotransferase II gene
  • aadA gene which confers spectinomycin and streptomycin resistance
  • phosphinothricin acetyl transferase bar gene
  • Ignite AgrEvo
  • Basta Hoechst
  • hpt hygromycin phosphotransferase gene
  • reporter genes coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/ or a visible signal (e.g., lucif erase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated.
  • the reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
  • Prunus Prunus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006 ;25(2):117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(l):38-45);
  • Example 1 Identification and isolation of toxin genes ftom thtee BtevibaciUus laterosporus strains
  • Table 1 List of source strains and deposits.
  • Genomes were first assembled using ABySS v.1.3.0 de Bruijn assembler and then further refined using the software Geneious® Pro 5.6.3 (Biomatters, http:/ /www. geneious. com/) and associated programmes.
  • a stand-alone BLASTX programme (NCBI) was used to integrate each partially assembled genome for the presence of potential toxin genes. Protein sequences of Cry, Cyt, binary, VIP and Isp genes were used, as well as other known bacterial insecticidal proteins. To rapidly identify any toxin encoding regions with homology known toxic proteins, genome sequence of isolate 1821 was performed, followed at a later date by genome sequencing of 1921 and RSP. The isolate 1821 was sequenced twice, independently.
  • Example 2 Demonsttating activity by expressing toxin ptoteins in E.coli and feeding to insects
  • Example 1 The individual polynculeotides/polypep tides described in Example 1 (and any associated open reading frames) can be expressed using commercially available non-conjugative vectors such as pET in E. coli (GATEWAY® technology, Invitrogen).
  • the transformed E. coli (as a bacterial cell in broth culture) can be used in a standard bioassay against diamondback moth (DBM) and other insects, to demonstrate insecticidal activity.
  • DBM diamondback moth
  • Such bioassays can also potentially determine the role of the gene in the observed virulence of the B. laterosporus isolates.
  • the kit uses Transform One Shot® Chemically Competent E. coli.
  • the pET vectors carry a bacteriophage T7 promotor, transcription and translation signals.
  • the source of T7 RNA polymerase is provided by the host cells.
  • Diamondback moth Plutella xylo Stella (Eepidoptera: Plutellidae), Cabbage white butterfly, Pieris rapae (Eepidoptera: Pieridae) and cabbage looper moth, Trichoplusia ni (Eepidoptera: Noctuidae)
  • Diamondback moth larvae can be reared on brassica (cabbage plants) at Lincoln, or the strains resistant to CrylA and CrylC and a susceptible (G88) strain can be obtained and tested at the New York State Agricultural Experiment Station, College of Agriculture and Life Sciences at Georgia University, located in Geneva, NY, USA. Cabbage white butterfly larvae can be field- collected from the farm at Lincoln University, New Zealand.
  • Ten 2 nd -3 ld instar larvae can be used and placed on 3cm disc of cabbage leaf treated with either 20 ⁇ of transformed E.coli solution, or dipped in the solution.
  • a wetting agent Siliwet L-77 (Momentive Performance Materials, New York, USA) or Triton X-100 (Rohm and Hass Co, Philadelphia, USA) is used at ⁇ 0.05%.
  • Each treatment can be replicated 3-5 times (3-50 larvae per treatment). Treated larvae remain on the cabbage leaf at 23°C 16L:8HD (Lincoln) or at 27°C 16hL:8hD (USA) and are checked daily for dead larvae.
  • Example 2 Demonstrating activity in transgenic plants and in plant bioassay
  • Sequences can be cloned into suitable constructs and vectors for transformation of plants as is well known by those skilled in the art, and disclosed herein.
  • the plant transformation vector, pHZBar is derived from pART27 (Gleave 1992, Plant Mol Biol 20: 1203—1207).
  • the pnos-nptII-nos3' selection cassette has been replaced by the CaMV35S-BAR-OCS3' selection cassette with the bar gene (which confers resistance to the herbicide ammonium glufosinate) expressed from the CaMV 35S promoter.
  • Cloning of expression cassettes into this binary vector is facilitated by a unique Notl restriction site and selection of recombinants by blue/white screening for ⁇ — galactosidase.
  • polynucleotide sequences of the invention can be cloned by standard techniques into pART7 downstream of the 35S promoter.
  • a unique Noil fragment can then be shuttled into pART27 (Gleave, 1992, Plant Mol Biol 20: 1203-1207) for transformation of various plant species.
  • This binary vector contains the nptll selection gene for kanamycin resistance under the control of the CaMV 35S promoter.
  • Genetic constructs in pART27 can be transferred into Agrobacterium tumefaciens strain GV3101 or EHA105 as plasmid DNA using freeze-thaw transformation method (Ditta et a/ 1980, Proc. Nad. Acad. Sci. USA 77: 7347-7351).
  • Agrobacterium The structure of the constructs maintained in Agrobacterium can be confirmed by restriction digest of plasmid DNA's prepared from bacterial culture. Agrobacterium cultures can be prepared in glycerol and transferred to -80°C for long term storage. Genetic constructs maintained in Agrobacterium strain GV3101 can be inoculated into 25 mL of MGL broth containing
  • Plants can be transformed to express the polynucleotides/polypeptides of the invention by numerous methods well known to those skilled in the art and disclosed herein.
  • Tobacco can be transformed via the leaf disk transformation-regeneration method (Horsch et al.1985).
  • Leaf disks from sterile wild type W38 tobacco plants are inoculated with an
  • Agrobactenum tumefaciens strain containing the appropriate binary vector, and cultured for 3 days.
  • the leaf disks are then transferred to MS selective medium containing 100 mg/L of kanamycin (or 5mg/L of glufosinate) and 300 mg/L of cefotaxime.
  • Shoot regeneration occurs over a month, and the leaf explants are placed on hormone free medium containing kanamycin or glufosinate for root formation.
  • constructs described above can also be used to transform Sorghum.
  • a suitable protocol for transforming Sorghum is found in Howe et al., 2006, Plant Cell Reports, Volume 25, No 8, 784- 791.
  • constructs described above can also be used to transform cotton.
  • a suitable protocol for transforming cotton is found in US 5, 846, 797.
  • constructs described above can also be used to transform mai ⁇ e.
  • a suitable protocol for transforming maize is found in US, 8,247, 369. Transformation of other plants
  • Transformation protocols for other plants are well-known to those skilled in the art, and are disclosed herein.
  • ELISA analysis according to the method disclosed in U.S. Pat. No. 5,625,136 can be used for the quantitative determination of the level of the toxin protein in transgenic plants, or parts thereof.
  • Various parts of the transformed plants, or whole plants can be used in standard insect bioassay procedures to test the activity of the polypeptides of the invention, and determine activity of the polypeptides, and the resistance of the transformed plants to various insects.

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Agronomy & Crop Science (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des polynucléotides isolés codant pour des polypeptides, des variants et fragments de ceux-ci ayant une activité insecticide. L'invention concerne également les polypeptides, variants et fragments correspondants. L'invention concerne des constructions, des cellules et des plantes contenant les polynucléotides, variants et fragments. L'invention concerne également des procédés de production de plantes résistantes à un insecte, et des procédés pour la lutte contre des nuisibles insectes à l'aide des polynucléotides and polypeptides de l'invention.
PCT/IB2013/061257 2012-12-24 2013-12-23 Polynucléotides, polypeptides et leurs procédés d'utilisation WO2014102697A2 (fr)

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WO2021040536A1 (fr) * 2019-08-23 2021-03-04 Ecolibrium Biologicals Holdings Limited Compositions de lutte biologique et utilisations associées

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US20070056061A1 (en) * 2000-05-18 2007-03-08 Bayer Bioscience N.V. Novel toxins
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021040537A1 (fr) * 2019-08-23 2021-03-04 Ecolibrium Biologicals Holdings Limited Polypeptides bioactifs et procédés associés
WO2021040536A1 (fr) * 2019-08-23 2021-03-04 Ecolibrium Biologicals Holdings Limited Compositions de lutte biologique et utilisations associées
GB2620001A (en) * 2019-08-23 2023-12-27 Cellora Ltd Bioactive polypeptides and methods related thereto

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