Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/18—Treatment of tobacco products or tobacco substitutes
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
  • A24B15/18—Treatment of tobacco products or tobacco substitutes
  • A24B15/24—Treatment of tobacco products or tobacco substitutes by extraction; Tobacco extracts
  • A24B15/241—Extraction of specific substances
  • A24B15/245—Nitrosamines
• • A—HUMAN NECESSITIES
  • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
  • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
  • A24B3/00—Preparing tobacco in the factory
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• polypeptide sequences of these genes are set forth in SEQ ID NOs: 1 -4, 10 and 1 1 and the polypeptide sequences of these genes are set forth in SEQ ID NOs: 5-7 and 12-14.
• the inventors unexpectedly found that a reduction in at least NNK is seen in cured plant material from both NtCLCe-RNAi and CLC-Nt2-RNAi plants. A reduction in total TSNA content was also observed. Reducing the expression of NtCLCe and/or CLC-Nt2 therefore contributes to reducing nitrate levels in tobacco leaves. After curing, at least NNK and optionally other TSNAs, which may include NNN or NAB or NAT or a combination of two or more thereof, can be reduced. In addition, the visual appearance of the plants is not substantially altered which is an important criterion for acceptance by the industry and for maximising plant yields and the like.
• nitrate content is about 6mg/g or less and the nicotine content is about 13 mg/g or less.
• construct, vector or expression vector comprising one or more of the isolated polynucleotide(s) described herein.
• Figure 4 Percentage of NNK in air-cured leaves of wt, NtCLCe-RNAi and CLC-Nt2-RNAi plants, after cultivation in 10 litre pots as shown in Figure 3. In this experiment, the highest NNK value corresponds to 108 ng/g.
• plant refers to any plant at any stage of its life cycle or development, and its progenies.
• the plant is a "tobacco plant”, which refers to a plant belonging to the genus Nicotiana. Preferred species of tobacco plant are described herein.
• reduce refers to a reduction of from about 10% to about 99%, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of a quantity or an activity, such as but not limited to polypeptide activity, transcriptional activity and protein expression.
• an isolated polynucleotide comprising, consisting or consisting essentially of a polynucleotide sequence having at least 60% sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence lisiting.
• the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.
• an isolated polynucleotide comprising, consisting or consisting essentially of a polynucleotide sequence having at least 60% sequence identity to SEQ ID N0.1 or SEQ ID NO:2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO:10 or SEQ ID NO:1 1 .
• a polynucleotide as described herein can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof.
• an isolated polypeptide comprising, consisting or consisting essentially of a polypeptide sequence having at least 60% sequence identity to any of the sequences described herein, including any of the polypeptides shown in the sequence lisiting.
• the isolated polypeptide comprises, consists or consists essentially of a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.
• SEQ ID NO:7 includes one or more mutations at amino acid positions selected from the group consisting of 21 , 58, 141 , 175, 5, 34, 124, 40, 8, 35, 30, 177, 42, 88, 155, 158, 170, 174, 126 or 131 or a combination of two or more thereof.
• the type of mutation(s) at this position can be a deletion, an insertion, a substitution or a missense mutation or a combination thereof.
• the mutation(s) can be a heterozygous or homozygous mutation, suitably, a homozygous mutation.
• the mutation(s) is a substitution mutation.
• the level of nitrate in the mutant plant is reduced to about 9 mg/g and the level of nitrate in the control plant increases to about 9 mg/g.
• the nitrate level in the control plant decreases.
• the level of nitrate decreases to about 2 mg/g in the mutant plant and decreases to about 4 mg/g in the control plant.
• the level of nictoine is somewhat similar during the morning for each of the mutant and control plants.
• the level of nictoine varies between aboutn 20 mg/g and about 24 mg/g for the mutant plant and about 15 mg/g abnd 17 mg/g for the control plant.
• the nictoine result indicates that the metabolism of the mutant plant is normal.
• the biomass levels for the mutant and the control plant are also comparable.
• non-naturally occurring as used herein describes an entity (for example, a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material) that is not formed by nature or that does not exist in nature.
• entity for example, a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material
• Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art.
• Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man.
• One of the strands of the small interfering RNA duplex can anneal to a complementary sequence within the target mRNA and related RNA variants.
• the small interfering RNA/mRNA duplexes are recognized by RNA-induced silencing complexes that can cleave RNAs at multiple sites in a sequence-dependent manner, resulting in the degradation of the target mRNA and related RNA variants.
• the nucleotides comprising the overhang can be ribonucleotides, deoxyribonucleotides or modified versions thereof.
• at least one strand of the interfering RNA molecule has a 3' overhang from about 1 to about 6 nucleotides in length.
• the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
• TILLING is another mutagenesis technology that can be used to generate and/or identify polynucleotides encoding polypeptides with modified expression and/or activity. TILLING also allows selection of plants carrying such mutants. TILLING combines high-density mutagenesis with high-throughput screening methods. Methods for TILLING are well known in the art (see McCallum et al., (2000) Nat Biotechnol 18: 455-457 and Stemple (2004) Nat Rev Genet 5(2): 145-50). Various embodiments are directed to expression vectors comprising one or more of the polynucleotides or interfering RNA constructs that comprise one or more polynucleotides described herein.
• Various embodiments are directed to expression vectors comprising one or more polynucleotides or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides described herein that are capable of self-annealing to form a hairpin structure, in which the construct comprises (a) one or more of the polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence comprising a reverse complementary sequence of the first sequence, positioned in the same orientation as the first sequence, wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.
• Various embodiments are directed to methods for modulating the expression level of one or more of the polynucleotide(s) described herein (or any combination thereof as described herein) by integrating multiple copies of the polynucleotide(s) into a (tobacco) plant genome, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to a polynucleotide.
• Mutations can result in homozygous disruption of one or more genes, in heterozygous disruption of one or more genes, or a combination of both homozygous and heterozygous disruptions if more than one gene is disrupted.
• Suitable transposable elements include retrotransposons, retroposons, and SINE-like elements. Such methods are known to persons skilled in the art.
• the mutant or non-naturally occurring plants may have one or more mutations in a region outside of one or more gene(s) - such as in a region upstream or downstream of the gene it regulates provided that they modulate the activity or expression of the gene(s).
• Upstream elements can include promoters, enhancers or transription factors. Some elements - such as enhancers - can be positioned upstream or downstream of the gene it regulates. The element(s) need not be located near to the gene that it regulates since some elements have been found located several hundred thousand base pairs upstream or downstream of the gene that it regulates.
• the seeds resulting from that pollination are grown into first generation plants. Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation.
• the treating step involves subjecting the at least one cell to a chemical mutagenising agent as descibed above and under conditions effective to yield at least one mutant plant cell.
• the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell.
• mutant plant includes mutants plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification.
• a zinc finger protein may be engineered to recognize a selected target site in a gene.
• a zinc finger protein can comprise any combination of motifs derived from natural zinc finger DNA-binding domains and non-natural zinc finger DNA-binding domains by truncation or expansion or a process of site-directed mutagenesis coupled to a selection method such as, but not limited to, phage display selection, bacterial two-hybrid selection or bacterial one-hybrid selection.
• the term "non- natural zinc finger DNA-binding domain” refers to a zinc finger DNA-binding domain that binds a three-base pair sequence within the target nucleic acid and that does not occur in the cell or organism comprising the nucleic acid which is to be modified.
• a zinc finger nuclease introduces a double stranded break in a regulatory region, a coding region, or a non-coding region of a genomic DNA sequence of a polynucleotide and leads to a reduction of the level of expression of a polynucleotide, or a reduction in the activity of the protein encoded thereby. Cleavage by zinc finger nucleases frequently results in the deletion of DNA at the cleavage site following DNA repair by nonhomologous end joining.
• a zinc finger protein may be selected to bind to a regulatory sequence of a polynucleotide. More specifically, the regulatory sequence may comprise a transcription initiation site, a start codon, a region of an exon, a boundary of an exon-intron, a terminator, or a stop codon. Accordingly, the invention provides a mutant, non-naturally occurring or transgenic plant or plant cells, produced by zinc finger nuclease-mediated mutagenesis in the vicinity of or within one or more polynucleotides described herein, and methods for making such a plant or plant cell by zinc finger nuclease-mediated mutagenesis. Methods for delivering zinc finger protein and zinc finger nuclease to a tobacco plant are similar to those described below for delivery of meganuclease.
• Plants suitable for use in genetic modification include, but are not limited to, monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae
• Minor compounds those typically found at significantly lower levels than the principal TSNAs, include 4-(methylnitrosamino) 4-(3-pyridyl)butanal (NNA), 4- (methylnitrosamino)-l -(3-pyridyl)-1 -butanol (NNAL), 4-(methylnitrosamino)4-(3-pyridyl)-1 -butanol (iso-NNAL), and 4-(methylnitrosamino)-4-(3-pyridyl)-1 -butyric acid (iso-NNAC).
• NNA 4-(methylnitrosamino) 4-(3-pyridyl)butanal
• NNA 4- (methylnitrosamino)-l -(3-pyridyl)-1 -butanol
• NAL 4-(methylnitrosamino)4-(3-pyridyl)-1 -butanol
• iso-NNAC 4-(methylnitrosamino)-4-
• a still further aspect relates to a cured plant material - such as cured leaf or cured tobacco - derived or derivable from a mutant, non-naturally occurring or transgenic plant or cell, wherein expression of one or more of the polynucleotides described herein or the activity of the protein encoded thereby is reduced and wherein the nitrate and/or NNK level is reduced by at least 5% as compared to a control plant.
• the level of NNN will be substantially the same.
• the total TSNA content is about 250 ng/g or less - such as about 240 ng/g or less, about 230 ng/g or less, about 220 ng/g or less, about 210 ng/g or less, about 200 ng/g or less, about 190 ng/g or less, about 180 ng/g or less, about 170 ng/g or less, about 160 ng/g or less, or about 150 ng/g or less.
• the total TSNA content is about 250 ng/g or less - such as about 240 ng/g or less, about 230 ng/g or less, about 220 ng/g or less, about 210 ng/g or less, about 200 ng/g or less, about 190 ng/g or less, about 180 ng/g or less, about 170 ng/g or less, about 160 ng/g or less, or about 150 ng/g or less.
• Another exemplary modification results in altered reproductive capability, such as male sterility.
• Another exemplary modification results in plants that are tolerant to abiotic stress (for example, drought, temperature, salinity), and tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol improve drought resistance.
• abiotic stress for example, drought, temperature, salinity
• Another exemplary modification results in plants that produce proteins which may have favourable immunogenic properties for use in humans.
• the invention also provides methods for producing seeds comprising cultivating the mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting seeds from the cultivated plants.
• Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
• Packaging material such as paper and cloth are well known in the art.
• a package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.
• Compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a polynucleotide (or any combination thereof as described herein) in a sample of polynucleotide.
• Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.
• the specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs).
• AFLPs are the product of allelic differences between amplification fragments caused by nucleotide sequence variability.
• the present invention further provides a means to follow segregation of one or more genes or nucleic acids as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as AFLP analysis.

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• 2013
  • 2013-12-19 JP JP2015548608A patent/JP6693747B2/ja active Active
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• 2015
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