WO2013107527A1 - Gènes nouveaux et utilisation de ces derniers pour la production de métabolites secondaires dans les plantes - Google Patents

Gènes nouveaux et utilisation de ces derniers pour la production de métabolites secondaires dans les plantes Download PDF

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WO2013107527A1
WO2013107527A1 PCT/EP2012/050969 EP2012050969W WO2013107527A1 WO 2013107527 A1 WO2013107527 A1 WO 2013107527A1 EP 2012050969 W EP2012050969 W EP 2012050969W WO 2013107527 A1 WO2013107527 A1 WO 2013107527A1
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plant
polypeptide
sequence
seq
polynucleotide
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PCT/EP2012/050969
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English (en)
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Alain Goossens
Miriam Onrubia IBAÑEZ
Javier Palazón BARANDELA
Rosa M. Cusidó VIDAL
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Vib Vzw
Universiteit Gent
University Of Barcelona
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Publication of WO2013107527A1 publication Critical patent/WO2013107527A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
    • 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

Definitions

  • the current invention relates to the field of secondary metabolite production in plants and plant cell cultures. More specifically, the present invention relates to the identification of novel genes involved in the biosynthesis and/or production of secondary metabolites in plants.
  • the plant kingdom produces tens of thousands of different small compounds with very complex structures that are often genus or family specific. These molecules, usually called secondary metabolites, display an immense variety in structures and biological activities that plants have tapped into over the course of evolution, and that is now harnessed by man for industrial and medical applications. These compounds play for example a role in the resistance against pests and diseases, attraction of pollinators and interaction with s ymbiotic microorganisms. Besides the importance for the plant itself, secondary metabolites are of great interest because they determine the quality of food (colour, taste, aroma) and ornamental plants (flower colour, smell). A number of secondary metabolites isolated from plants are commercially available as fine chemicals, for example, drugs, dyes, flavours, fragrances and even pesticides.
  • taxol is a highly substituted, polyoxygenated cyclic diterpenoid characterised by the taxane ring system, which presents an excellent antitumoral activity against a range of cancers.
  • the cellular and genetic programs that steer the production of secondary metabolites can be launched rapidly when plants perceive particular environmental stimuli.
  • the jasmonate phytohormones (JAs) play a prominent and universal role in mediating these responses as they can induce synthetic pathways of molecules of a wide structural variety, encompassing all major secondary metabolites (Zhao et al. 2005; Pauwels et al. 2009).
  • the present invention provides a regulatory gene encoding a peptide which can be used to induce or enhance the production and/or secretion of secondary metabolites in plants or cells derived thereof. More specifically, the regulatory gene of the present invention codes for a plant peptide that synergistically interacts with elicitors of secondary metabolism. According to one aspect, the invention relates to an isolated polypeptide selected from the group consisting of:
  • polypeptide comprising a polypeptide sequence having a least 75% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1;
  • polypeptide comprising an amino acid sequence with at least 75% identity to SEQ ID NO: 2;
  • the invention relates to any of the above described polypeptides wherein said polypeptide sequence is consisting of an amino acid sequence as set forth in SEQ ID NO: 2 or 3 and polypeptide sequences having at least 75% identity to SEQ ID NO: 2 or 3.
  • the invention relates to an isolated polynucleotide selected from the group consisting of:
  • polynucleotide comprising a polynucleotide sequence having at least 70% identity to the sequence having SEQ ID NO: 1;
  • the invention in another embodiment, relates to a chimeric gene comprising the following operably- linked sequences: a) a promoter region capable of directing expression in a plant or plant cell; b) a DNA region encoding a polypeptide as defined above; c) a 3' polyadenylation and transcript termination region.
  • a vector comprising a polynucleotide sequence or a chimeric gene as defined above also forms part of the present invention, as well as a host cell comprising a polynucleotide sequence or a chimeric gene or a vector as defined above.
  • a further aspect of the present invention relates to a transgenic plant or a cell derived thereof that is transformed with a vector or that comprises a chimeric gene as defined above.
  • Said transgenic plant or cell derived thereof can be from the group comprising Taxus spp., Medicago spp., Catharanthus spp., Nicotiana spp., Artemisia spp., or any other plant species.
  • the above described polynucleotide sequences can be used for the enhanced production and/or stimulation of biosynthesis of secondary metabolites in plants or plant cells.
  • Said secondary metabolites comprise alkaloid compounds, phenylpropanoid compounds or terpenoid compounds.
  • FIGURE 1 Average linkage hierarchical clustering of MeJA-modulated gene tags identified by cDNA-AFLP of elicited Taxus baccata cells.
  • the time points (h) are indicated at the top.
  • Red and green boxes reflect transcriptional activation and repression, respectively, relative to the average expression level in control (DMSO) cells. Gray boxes correspond to missing time points.
  • OB NtORCl+NbbHLHl
  • FIGURE 4 Total alkaloid accumulation in transgenic Nicotiana tabacum hairy roots after 8 days of elicitation. Values are expressed as ⁇ g/g DW and compare hairy roots transformed with TB595 or control vector. EtOH: control; MeJ: Methyl jasmonate treatment. Alkaloids correspond to: green, Nicotine; yellow, Anabasine; orange, Anatabine. Values are mean ⁇ SE of 3-4 independent samples.
  • FIGURE 5 The TB595 peptide sequence is highly conserved in the plant kingdom. Multiple sequence alignment generated with ClustalW of the TB595 homologous sequences of Arabidopsis thaliana (At), Oryza sativa (Os), Picea sitchensis (Picea), Physcomitrella patens (Pp), Ricinus communis (Rc) and Selaginella moellendorffi (Sm).
  • FIGURE 6 Phylogenetic tree of TB595 orthologs in A.thaliana and M. truncatula.
  • FIGURE 7 Signal peptide prediction with SignalP in the TB595 sequence.
  • FIGURE 8 The TB595 peptide sequence.
  • the signal peptide sequence is underlined, the mature peptide in black and bold.
  • the present invention provides a regulatory gene which can be used to modulate the production of secondary metabolites in organisms and cells derived thereof. It was surprisingly found that the regulatory gene of the present invention codes for a small plant peptide hormone, including an N-terminal signal peptide, that synergistically interacts with elicitors of secondary metabolism.
  • the invention relates to an isolated polypeptide selected from the group consisting of:
  • polypeptide comprising a polypeptide sequence having a least 75% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1;
  • polypeptide comprising an amino acid sequence with at least 75% identity to SEQ ID NO: 2;
  • the invention relates to any of the above isolated polypeptides wherein said polypeptide sequence is consisting of an amino acid sequence as set forth in SEQ ID NO: 2 or 3 and polypeptide sequences having at least 75% identity to SEQ ID NO: 2 or 3.
  • the invention provides for an isolated polynucleotide selected from the group consisting of:
  • polynucleotide comprising a polynucleotide sequence having at least 70% identity to the sequence having SEQ ID NO: 1;
  • polypeptide As used herein, the terms “polypeptide”, “protein”, “peptide” are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • nucleic acid As used herein, the terms “nucleic acid”, “polynucleotide”, “polynucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger NA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the polynucleotide molecule may be linear or circular.
  • the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like.
  • the polynucleotide may comprise single stranded or double stranded DNA or RNA.
  • the polynucleotide may comprise modified bases or a modified backbone.
  • a nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.
  • isolated polypeptide or an “isolated polynucleotide”, as used herein, refers to respectively an amino acid sequence or a polynucleotide sequence which is not naturally-occurring or no longer occurring in the natural environment wherein it was originally present.
  • the terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 75% identity over a specified region) when compared and align ed for maximum correspondence over a comparison window or designated region as measured using sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides or even more in length.
  • the invention relates to an isolated polypeptide comprising a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the polypeptide encoded by a polynucleotide sequence having SEQ ID NO: 1 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2 or 3.
  • the invention relates to an isolated polynucleotide comprising a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence having SEQ ID NO: 1.
  • fragments and variants of any of the above polynucleotides or polypeptides also from part of the present invention.
  • a fragment refers to any sequence of at least 15 consecutive nucleotides, preferably at least 30 consecutive nucleotides, more preferably at least 50, 60, 70, 80, 90, 100, 150, 200 consecutive nucleotides or more, of any of the sequences provided herein. If desired, the fragment may be fused at either terminus to additional base pairs, which may number from 1 to 20, typically 50 to 100, but up to 250 to 500 or more.
  • a “functional fragment” means a polypeptide fragment possessing the biological property able to modulate the production of secondary metabolites (as described further herein) in an organism or cell derived thereof. In a particular embodiment said functional fragment is able to modulate the production of a secondary metabolite in a plant or plant cell derived thereof.
  • a fragment may comprise, for example, an activation domain or a domain for protein-protein interactions. In a particular embodiment, a fragment may comprise a receptor binding domain.
  • a “variant” as used herein refers to homologs, orthologs and paralogs and include, but are not limited to, homologs, orthologs and paralogs of SEQ. ID NOs: 1, 2 or 3.
  • Homologs of a protein encompass peptides, oligopeptides and polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity.
  • Orthologs and paralogs which are well-known terms by the skilled person, define subcategories of homologs and encompass evolutionary concepts used to describe the ancestral relationships of genes.
  • Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
  • General methods for identifying orthologues and paralogues include phylogenetic methods, sequence similarity and hybridization methods. Percentage similarity and identity can be determined electronically. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990).
  • said homologue, orthologue or paralogue has a sequence identity at protein level of at least 50%, preferably 60%, more preferably 70%, even more preferably 80%, most preferably 90% as measured in a BLASTp.
  • non-limiting examples of functionally homologous sequences of TB595 include At2g20562, At2g31090 (Arabidopsis thaliana); Os05g0209500, Os01g0214500 (Oryza sativa); ABK22794, ABK23667 (Picea sitchensis); XP_001756953, XP_001754417, XP_001756644 (Physcomitrella patens); XP_002522544, XP_002517854 (Ricinus communis); XP_002967828 (Selaginella moellendorffi); XP_002309758, XP_002328037, XP_002317478, XP_002329114, XP_002330749, XP_002326022, XP_002317432 (Populus trichocarpa); ACU14314,
  • said variants have an increased activity.
  • any of a variety of polynucleotide sequences are capable of encoding the polypeptides of the present invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides. Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed "silent" variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art.
  • any and all such variations of a sequence are a feature of the invention.
  • other conservative variations that alter one, or a few amino acids in the encoded polypeptide can be made without altering the function of the polypeptide (i.e. enhanced secondary metabolite production, in the context of the present invention), these conservative variants are, likewise, a feature of the invention.
  • Substitutions that are less conservative than those in Table 1 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
  • a hydrophilic residue e.g
  • substitutions, deletions and insertions introduced into the sequences are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis or the other methods known in the art. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotides of the invention should not create complementary regions that could produce secondary m NA structure.
  • the polypeptide encoded by the DNA performs the desired function (i.e. enhanced secondary metabolite production, in the context of the present invention).
  • the invention also relates to a chimeric gene, or otherwise expression cassette, comprising the following operably-linked sequences: a) a promoter region capable of directing expression in a host organism; b) a DNA region encoding a polypeptide according to any of claims 1 or 2; c) a 3' polyadenylation and transcript termination region.
  • host organism is understood as being any unicellular or multicellular organism into which the chimeric gene according to the invention can be introduced for the purpose of producing secondary metabolites (as described further herein).
  • These organisms are, in particular, bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces, Kluyveromyces, Pichia, Yarrowia, Hansenula, Kogamataella, fungi, in particular Aspergillus, a baculovirus, algae, animal cells, or, preferably, plant cells and plants.
  • Plant cell is understood, according to the invention, as being any cell which is derived from or found in a plant and which is able to form or is part of undifferentiated tissues, such as calli, differentiated tissues such as embryos, parts of plants, plants or seeds.
  • operably linked refers to a linkage in which the regulatory sequence is contiguous with the gene of interest to control the gene of interest, as well as regulatory sequences that act in trans or at a distance to control the gene of interest.
  • a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence.
  • a DNA for a signal sequence is operably linked to DNA coding for a polypeptide if it is expressed as a pre-protein that participates in the transport of the polypeptide.
  • regulatory sequence refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked.
  • Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient NA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRMA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • the invention provides for a vector comprising any of the above described polynucleotide sequences or chimeric gene. Further a host cell comprising any of the above described polynucleotides or chimeric gene or vector are also envisaged herein.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • the vector may be of any suitable type including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • certain preferred vectors are capable of directing the expression of certain genes of interest.
  • recombinant expression vectors Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g. plant cell). Typically, a recombinant vector according to the present invention comprises at least one "chimeric gene” or "expression cassette". Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof of the present invention operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal.
  • the promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell.
  • recombinant host cell ("expression host cell”, “expression host system”, “expression system” or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. Host cells can be of bacterial, fungal, plant or mammalian origin.
  • the invention provides a transgenic plant or a cell derived thereof that is transformed with the above described vector.
  • plant refers to vascular plants (e.g. gymnosperms and angiosperms).
  • a "transgenic plant” refers to a plant comprising a recombinant polynucleotide and/or a recombinant polypeptide according to the invention.
  • a transgenic plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, and progeny thereof.
  • a transgenic plant can be obtained by transforming a plant cell with an expression cassette of the present invention and regenerating such plant cell into a transgenic plant. Such plants can be propagated vegetatively or reproductively.
  • the transforming step may be carried out by any suitable means, including by Agrobacterium-mediated transformation and non-zAgrofaocier/um-mediated transformation, as discussed in detail below.
  • Plants can be regenerated from the transformed cell (or cells) by techniques known to those skilled in the art. Where chimeric plants are produced by the process, plants in which all cells are transformed may be regenerated from chimeric plants having transformed germ cells, as is known in the art.
  • Methods that can be used to transform plant cells or tissue with expression vectors of the present invention include both Agrobacterium and non- Agrobacterium vectors.
  • Agrobacterium-mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes and is described in detail in Gheysen, G., Angenon, G. and Van Montagu, M. 1998. Agrobacterium-mediated plant transformation: a scientifically interesting story with significant applications.
  • K. Lindsey (Ed.), Transgenic Plant Research. Harwood Academic Publishers, Amsterdam, pp. 1-33 and in Stafford, H.A. (2000) Botanical Review 66: 99-118.
  • a second group of transformation methods is the non-Agrobacterium mediated transformation and these methods are known as direct gene transfer methods.
  • An overview is brought by Barcelo, P. and Lazzeri, P. A. (1998) Direct gene transfer: chemical, electrical and physical methods.
  • K. Lindsey (Ed.), Transgenic Plant Research, Harwood Academic Publishers, Amsterdam, pp.35-55.
  • Methods include particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome- mediated transformation, silicon-whiskers mediated transformation etc.
  • Hairy root cultures can be obtained by transformation with virulent strains of Agrobacterium rhizogenes, and they can produce high contents of secondary metabolites characteristic to the mother plant. Protocols used for establishing of hairy root cultures vary, as well as the susceptibility of plant species to infection by Agrobacterium (Toivounen et al. 1993; Vanhala et al. 1995). It is known that the Agrobacterium strain used for transformation has a great influence on root morphology and the degree of secondary metabolite accumulation in hairy root cultures. It is possible by systematic clone selection e.g. via protoplasts, to find high yielding, stable, and from single cell derived-hairy root clones. This is possible because the hairy root cultures possess a great somaclonal variation. Another possibility of transformation is the use of viral vectors (Turpen 1999).
  • Any plant tissue or plant cells capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with an expression vector of the present invention.
  • organogenesis' means a process by which shoots and roots are developed sequentially from meristematic centers; the term 'embryogenesis' means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include protoplasts, leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyls meristem).
  • existing meristematic tissue e.g. apical meristems, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyls meristem.
  • control plant refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying a difference in production of secondary metabolite (as described further herein) in the transgenic or genetically modified plant.
  • a control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated.
  • a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested.
  • a suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line (wild type) used to generate a transgenic plant herein.
  • Plants of the present invention may include, but not limited to, plants or plant cells of agronomically important crops which are or are not intended for animal or human nutrition, such as maize or corn, wheat, barley, oat, Brassica spp. plants such as Brassica napus or Brassica juncea, soybean, bean, alfalfa, pea, rice, sugarcane, beetroot, tobacco, sunflower, cotton, Arabidopsis, vegetable plants such as cucumber, leek, carrot, tomato, lettuce, peppers, melon, watermelon, diverse herbs such as oregano, basilicum and mint. It may also be applied to plants that produce valuable compounds, e.g.
  • ajmalicine useful as for instance pharmaceuticals, as ajmalicine, vinblastine, vincristine, ajmaline, reserpine, rescinnamine, camptothecine, ellipticine, quinine, and quinidine, taxol, morphine, scopolamine, atropine, cocaine, sanguinarine, codeine, genistein, daidzein, digoxin, calystegins or as food additives such as anthocyanins, vanillin; including but not limited to the classes of compounds mentioned above.
  • Examples of such plants include, but not limited to, Papaver spp., Rauwolfia spp., Taxus spp., Cinchona spp., Eschscholtzia californica, Camptotheca acuminata, Hyoscyamus spp., Berberis spp., Coptis spp., Datura spp., Atropa spp., Thalictrum spp., Peganum spp.
  • Preferred members of the genus Taxus comprise Taxus brevifolia, Taxus baccata, Taxus cuspidate, Taxus Canadensis and Taxus floridana.
  • the polynucleotide sequence, homologue, variant or fragment thereof of the invention may be expressed in for example a plant cell under the control of a promoter that directs constitutive expression or regulated expression.
  • Regulated expression comprises temporally or spatially regulated expression and any other form of inducible or repressible expression.
  • Temporally means that the expression is induced at a certain time point, for instance, when a certain growth rate of the plant cell culture is obtained (e.g. the promoter is induced only in the stationary phase or at a certain stage of development).
  • Spatially means that the promoter is only active in specific organs, tissues, or cells (e.g. only in roots, leaves, epidermis, guard cells or the like).
  • regulated expression comprise promoters whose activity is induced or repressed by adding chemical or physical stimuli to the plant cell.
  • the expression is under control of environmental, hormonal, chemical, and/or developmental signals.
  • promoters for plant cells include promoters that are regulated by (1) heat, (2) light, (3) hormones, such as abscisic acid and methyl jasmonate (4) wounding or (5) chemicals such as salicylic acid, chitosans or metals. Indeed, it is well known that the expression of secondary metabolites can be boosted by the addition of for example specific chemicals, jasmonate and elicitors.
  • the co-expression of several (more than one) polynucleotide sequence or homologue or variant or fragment thereof, in combination with the induction of secondary metabolite synthesis is beneficial for an optimal and enhanced production of secondary metabolites.
  • the at least one polynucleotide sequence, homologue, variant or fragment thereof is placed under the control of a constitutive promoter.
  • a constitutive promoter directs expression in a wide range of cells under a wide range of conditions.
  • constitutive plant promoters useful for expressing heterologous polypeptides in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues including monocots; the nopaline synthase promoter and the octopine synthase promoter.
  • the expression cassette is usually provided in a DNA or NA construct which is typically called an "expression vector" which is any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide replication within a cell (i.e.
  • Suitable vectors include, but are not limited to, plasmids, bacteriophages, cosmids, plant viruses and artificial chromosomes.
  • the expression cassette may be provided in a DNA construct which also has at least one replication system. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts.
  • the markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant.
  • a biocide such as antibiotics, toxins, heavy metals, certain sugars or the like
  • b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant.
  • exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene.
  • markers are ⁇ -glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
  • promoter activity refers to the extent of transcription of a polynucleotide sequence, homologue, variant or fragment thereof that is operably linked to the promoter whose promoter activity is being measured.
  • the promoter activity may be measured directly by measuring the amount of RNA transcript produced, for example by Northern blot or indirectly by measuring the product coded for by the RNA transcript, such as when a reporter gene is linked to the promoter.
  • the above described polynucleotide sequences (and encoded proteins) can be used for inducing or enhancing the production of secondary metabolites and/or for the stimulation of biosynthesis of secondary metabolites in any organism wherein said organisms can be selected from the list comprising bacteria, for example E.
  • the present invention provides methods for inducing or enhancing the production of secondary metabolites chosen from the group comprising alkaloid compounds, phenylpropanoid compounds and terpenoid compounds, for which non-limiting examples are provided further herein.
  • the present invention provides methods for inducing or enhancing the production of at least one secondary metabolite, which is meant to include related structures of secondary metabolites and intermediates or precursors thereof.
  • a primary metabolite is any intermediate in, or product of the primary metabolism in cells.
  • the primary metabolism in cells is the sum of metabolic activities that are common to most, if not all, living cells and are necessary for basal growth and maintenance of the cells.
  • Primary metabolism thus includes pathways for generally modifying and synthesising certain carbohydrates, amino acids, fats and nucleic acids, with the compounds involved in the pathways being designated primary metabolites.
  • secondary metabolites usually do not appear to participate directly in growth and development.
  • Secondary plant metabolites include e. g. alkaloid compounds (e. g. terpenoid indole alkaloid, tropane alkaloid, steroid alkaloids), phenylpropanoid compounds (e. g. quinines, lignans and flavonoids), terpenoid compounds (e. g. monoterpenoids, iridoids, sesquiterpenoids, diterpenoids and triterpenoids).
  • secondary metabolites include small molecules, such as substituted heterocyclic compounds which may be monocyclic or polycyclic, fused or bridged.
  • plant secondary metabolites have value as pharmaceuticals.
  • Non-limiting examples of plant pharmaceuticals include e. g. taxol, digoxin, atropine, scopolamine, colchicine, diosgenin, codeine, cocaine, morphine, quinine, shikonin, ajmaline, vinblastine, vincristine, and others.
  • alkaloids refers to all nitrogen-containing natural products which are not otherwise classified as amino acid peptides and proteins, amines, cyanogenic glycosides, glucosinolates, antibiotics, phytohormones or primary metabolites (such as purine and pyrimidine bases).
  • Alkaloids can be divided into the following major groups: (i) "true alkaloids”, which contain nitrogen in the heterocycle and originate from amino acids, for which atropine, nicotine and morphine are characteristic examples; (ii) “protoalkaloids”, which contain nitrogen in the side chain and also originate from amino acids, for which mescaline, adrenaline and ephedrine are characteristic examples; (iii) "polyamine alkaloids” which are derivatives of putrescine, spermidine and spermine; (iv) “peptide and cyclopeptide alkaloids”; (v) “pseudoalkaloids”, which are alkaloid-like compounds which do not originate from amino acids, including terpene-like and steroid-like alkaloids as well as purine-like alkaloids such as caffeine, theobromine and thephylline.
  • Plants synthesize alkaloids for various defence-related reactions, e. g. actions against pathogens or herbivores. Enzymes and genes have been partly characterised only in groups of nicotine and tropane alkaloids, indole alkaloids and isoquinolidine alkaloids (Suzuki et al. 1999). Nicotine and tropane alkaloid share partly the same biosynthetic pathway. Many plants belonging to, for example, the Solanaceae family, have been used for centuries because of their active substances hyoscyamine and scopolamine. Also other Solanaceae plants belonging to the genera Atropa, Datura, Duboisia and Scopolia produce these valuable alkaloids.
  • the "calystegins” constitute a unique subgroup of the tropane alkaloid class (Goldmann et al. 1990). They are characterized by the absence of an N-methyl substituent and a high degree of hydroxylation. Trihydroxylated calystegins are summarized as the calystegin A-group, tetrahydroxylated calystegins as the B-group, and pentahydroxylated derivates form the C-group.
  • Calystegins represent a novel structural class of tropane alkaloids possessing potent glycosidase inhibitory properties next to longer known classes of the monocyclic pyrrolidines (e. g. dihydroxymethyldihydroxy pyrrolidine), pyrrolines and piperidines (e. g.
  • Glycosidase inhibitors are potentially useful as antidiabetic, antiviral, antimetastatic, and immunomodulatory agents.
  • phenylpropanoids or "phenylpropanes”, refer to aromatic compounds with a propyl side-chain attached to the aromatic ring, which can be derived directly from phenylalanine.
  • the ring often carries oxygenated substituents (hydroxyl, methoxy and methylenedioxy groups) in the para- position.
  • Natural products in which the side-chain has been shortened or removed can also be derived from typical phenylpropanes.
  • Phenylpropanoids are found throughout the plant kingdom. Most plant phenolics are derived from the phenylpropanoid and phenylpropanoid-acetate pathways and fulfil a very broad range of physiological roles in plants. For example, polymeric lignins reinforce specialized cell wall.
  • Lignans which vary from dimers to higher oligomers. Lignans can either help defend against various pathogens or act as antioxidants in flowers, leaves and roots.
  • the flavonoids comprise an astonishingly diverse group of more than 4500 known compounds. Among their subclasses are the anthocyanins (pigments), proanthocyanidins or condensed tannins (feeding deterrents and wood protectants), and isoflavonoids (defensive products and signaling molecules).
  • the coumarins, furanocoumarins, and stilbenes protect against bacterial and fungal pathogens, discourage herbivory, and inhibit seed germination.
  • terpenoids or otherwise “isoprenoids” refer to the large and diverse class of naturally-occuring organic chemicals similar to terpenes and can be found in all classes of living organisms. Terpenoids are molecules derived from a five-carbon isoprene unit that are assembled and modified in different ways and have diverse activities. Plant terpenoids are used extensively for their aromatic qualities and contribute to e.g. the scent of eucalyptus, the flavors of cinnamon, clover and ginger, and the color of yellow flowers. They play a role in traditional herbal remedies and may have antibacterial, antineoplastic, and other pharmaceutical functions.
  • terpenoids include citral, menthol, camphor, salvinorin A and cannabinoids and are also used to flavour and/or scent a variety of commercial products.
  • the steroids and sterols in animals are biologically produced from terpenoid precursors. They also include pharmaceuticals e.g. taxol, artemisinin.
  • Terpenoids are classified with reference to the number of isoprene units that comprise the particular terpenoid. For example a monoterpenoid comprises two isoprene units; a sesquiterpenoid comprises three isoprene units and a diterpenoid four isoprene units. Polyterpenoids comprise multiple isoprene units. There are many thousands of examples of terpenoids. Artemisinin is a sesquiterpene lactone endoperoxide and is a natural product produced by the plant Artemisia annua.
  • Artemisinin is typically used in combination with anti-malarial therapeutics, for example lumefantrine, mefloquine, amodiaquine, sulfadoxine, chloroquine, in artemisinin combination therapies (ACT).
  • Endoperoxides like artemisinin, for example dihydroartemisinin, artemether, and sodium artesunate have been used in the treatment of malaria.
  • monoterpenoids include linalool, citronellol, menthol, geraniol and terpineol. Linalool and citronellol are used as a scent in soap, detergents, shampoo and lotions. Linalool is also an intermediate in the synthesis of vitamin E.
  • Menthol is isolated from peppermint or other mint oils and is known for its anaesthetic properties; it is often included sore throat medications and oral medications e.g. for the treatment of bad breath in toothpaste and mouth wash.
  • Geraniol is known for its insect repellent properties and and is also used as a scent in perfumes.
  • Terpineol is also used as an ingredient in perfumes and cosmetics and as flavouring. It is apparent that in addition to the pharmaceutical applications of monoterpenoids such as perillyl alcohol there are additional uses as scents, flavourings and as insect deterrents.
  • the synthesis of terpenoids involves a large number of enzymes with different activities.
  • isoprene units are synthesized from monosaturated isoprene units by prenyltransferases into multiples of 2, 3 or 4 isoprene units. These molecules serve as substrates for terpene synthase enzymes, also called terpene cyclase. Plant terpene synthases are known in the art.
  • Taxanes are diterpenens which were originally described in the genes Taxus (yews).
  • taxoids is equivalent with the word “taxanes”.
  • taxol is a highly substituted, polyoxygenated cyclic diterpenoid characterised by the taxane ring system, which presents an excellent antitumoral activity against a range of cancers.
  • Paclitaxel is the generic name for taxol, which is a registered trademark of Bristol-Myers Squibb.
  • the taxol biosynthetic pathway is considered to require 19 enzymatic steps from the universal diterpenoid precursor geranylgeranyl diphosphate which is cyclised, in the commited step to taxa-4(5),ll(12)-diene.
  • a decrease of a secondary metabolite can for example refer to the decrease of an undesired intermediate product.
  • an increase in the production of one or more metabolites it is understood that said production may be enhanced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% relative to the untransformed organism (e.g. a plant or plant cell) which was used to transform with an expression vector comprising an expression cassette further comprising at least one polynucleotide or homologue or variant or fragment thereof of the invention.
  • a decrease in the production of the level of a secondary metabolite may be decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% relative to the untransformed organism (e.g. plant or plant cell) which was used to transform with an expression vector comprising an expression cassette further comprising at least one polynucleotide or homologue or variant or fragment thereof of the invention.
  • untransformed organism e.g. plant or plant cell
  • An "induced production" of a secondary metabolite means that there is no detectable production of secondary metabolite(s) by the untransformed plant (cell) but that detection becomes possible upon carrying out the transformation according to the invention.
  • An "enhanced production" of a secondary metabolite means that there exists already a detectable amount of secondary metabolite(s) by the untransformed plant cell but that detection becomes possible upon carrying out the transformation according to the invention and that an increase of secondary metabolite(s) can be measured by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more then 90% compared to basal secretion by the untransformed plant (cell).
  • the organism (such as a plant or plant cell line) transformed with a polynucleotide of the present invention is induced before it produces secondary metabolites.
  • the wording "inducing the production” means that for example the cell culture, such as a plant cell culture, is stimulated by the addition of an external factor. External factors include the application of heat, the application of cold, the addition of acids, bases, metal ions, fungal membrane proteins, sugars and the like.
  • External factors include the application of heat, the application of cold, the addition of acids, bases, metal ions, fungal membrane proteins, sugars and the like.
  • Elicitors are compounds capable of inducing defence responses in plants. These are usually not found in intact plants but their biosynthesis is induced after wounding or stress conditions.
  • Jasmonates are linoleic acid derivatives of the plasma membrane and display a wide distribution in the plant kingdom. They were originally classified as growth inhibitors or promoters of senescence but now it has become apparent that they have pleiotropic effects on plant growth and development. Jasmonates appear to regulate cell division, cell elongation and cell expansion and thereby stimulate organ or tissue formation. They are also involved in the signal transduction cascades that are activated by stress situations such as wounding, osmotic stress, desiccation and pathogen attack.
  • Methyl jasmonate is known to induce the accumulation of numerous defence-related secondary metabolites (e.g. phenolics, alkaloids and sesquiterpenes) through the induction of genes coding for the enzymes involved in the biosynthesis of these compounds in plants. Jasmonates can modulate gene expression from the (post)transcriptional to the (post)translational level, both in a positive as in a negative way. Genes that are upregulated are e.g. defence and stress related genes (PR proteins and enzymes involved with the synthesis of phytoalexins and other secondary metabolites) whereas the activity of housekeeping proteins and genes involved with photosynthetic carbon assimilation are down-regulated.
  • defence and stress related genes PR proteins and enzymes involved with the synthesis of phytoalexins and other secondary metabolites
  • the biosynthesis of phytoalexins and other secondary products in plants can also be boosted up by signal molecules derived from micro-organisms or plants (such as peptides, oligosaccharides, glycopeptides, salicylic acid and lipophilic substances) as well as by various abiotic elicitors like UV-light, heavy metals (Cu, VOS04, Cd) and ethylene.
  • signal molecules derived from micro-organisms or plants such as peptides, oligosaccharides, glycopeptides, salicylic acid and lipophilic substances
  • various abiotic elicitors like UV-light, heavy metals (Cu, VOS04, Cd) and ethylene.
  • the effect of any elicitor is dependent on a number of factors, such as the specificity of an elicitor, elicitor concentration, the duration of the treatment and growth stage of the culture.
  • secondary metabolites can be measured intracellularly or in the extracellular space by methods known in the art. Such methods comprise analysis by thin-layer chromatography, high pressure liquid chromatography, capillary electrophoresis, gas chromatography combined with mass spectrometric detection, radioimmuno-assay ( IA) and enzyme immuno-assay (ELISA).
  • IA radioimmuno-assay
  • ELISA enzyme immuno-assay
  • taxol and taxanes can be measured as described in Ketchum et al. (2007); tobacco alkaloid content can be analysed by GC-MS (Millet et al. 2009); Medicago flavonoid and triterpene saponin content can be analysed by Reversed phase UPLC/ICR/FT-MS (see for example as described in Example section).
  • the invention can be practiced with any plant variety for which cells of the plant can be transformed with an expression cassette of the current invention and for which transformed cells can be cultured in vitro.
  • Suspension culture, callus culture, hairy root culture, shoot culture or other conventional plant cell culture methods may be used (as described in: Drugs of Natural Origin, G. Samuelsson, 1999, ISBN 9186274813).
  • plant cells it is understood any cell which is derived from a plant and can be subsequently propagated as callus, plant cells in suspension, organized tissue and organs (e.g. hairy roots). Tissue cultures derived from the plant tissue of interest can be established.
  • Suitable culture media for callus induction and subsequent growth on aqueous or solidified media are known.
  • Exemplary media include standard growth media, many of which are commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt (SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, and other variations and supplements well known to those of skill in the art (see, e.g., Plant Cell Culture, Dixon, ed. I RL Press, Ltd.
  • SH Schenk-Hildebrandt
  • LS Linsmaier-Skoog
  • MS Murashige and Skoog
  • Gamborg's B5 medium Nitsch & Nitsch medium
  • White's medium and other variations and supplements well known to those of skill in the art (see, e.g., Plant Cell Culture, Dixon, e
  • suitable media include 1/2 MS, 1/2 L.P., DCR, Woody Plant Medium (WPM), Gamborg's B5 and its modifications, DV (Durzan and Ventimiglia, In Vitro Cell Dev. Biol. 30:219-227 (1994)), SH, and White's medium.
  • the invention envisages a method for the production or for stimulating the biosynthesis of secondary metabolites in plant or plant cell comprising the step of contacting said plant or plant cell derived thereof with a polypeptide of the present invention.
  • the present invention also relates to a method for the production or for stimulating the biosynthesis of secondary metabolites in a plant or a plant cell comprising the steps of transforming said plant or plant cell with a vector of the present invention or with a chimeric gene of the present invention and allowing the plant or the plant cell to grow.
  • the current invention can be combined with other known methods to enhance the production and/or the secretion of secondary metabolites in plant cell cultures such as (1) by improvement of the plant cell culture conditions, (2) by the transformation of the plant cells with a transcription factor capable of upregulating genes involved in the pathway of secondary metabolite formation, (3) by the addition of specific elicitors to the plant cell culture, and 4) by the induction of organogenesis.
  • the polynucleotides and polypeptides of the present invention can be used in all types of plants to boost the plant's own secondary metabolite production or in plants that are transformed with a combination of genetic material that can lead to the generation of novel metabolic pathways (for example through the interaction with metabolic pathways resident in the host organism or alternatively silent metabolic pathways can be unmasked) and eventually lead to the production of novel classes of compounds.
  • This novel or reconstituted metabolic pathways can have utility in the commercial production of novel, valuable compounds.
  • tag sequences have been obtained, almost all representing novel Taxus sequences. For 400 of these tag sequences a hit in the public database, and thus an indication on the possible function or activity was encountered.
  • 8 of the known Taxol biosynthesis genes (Croteau et al., 2006) are present, confirming the value and relevance of the sequence platform (Fig. 1).
  • full-length open reading frame sequences have been isolated for genes encoding the Jasmonate ZIM-domain (JAZ) transcription factors, known JA-inducible repressor proteins involved in JA signaling (Chico et al., 2008), indicating that the sequence platform also contains regulatory genes, of JA signaling in general and potentially of taxol biosynthesis in particular (Fig. 1).
  • One such gene (SEQ ID NO: 1; TB595 in Fig. 1) encodes for a hypothetical protein that potentially encodes a peptide hormone that may regulate taxol biosynthesis in planta and in vitro cultures.
  • EXAM PLE Functional analysis of the TB595 gene by transient expression in plant protoplasts with Taxus taxol synthesis reporter genes.
  • FL full-length cDNA clone corresponding to the short sequence TB595 tag isolated in the cDNA-AFLP analysis
  • the use of gene-specific primers, RT-PCR, 5'- and 3'-RACE (InVitroGen Life Technologies) techniques were combined to yield a FL TB595 cDNA clone (SEQ ID NO: 1).
  • the corresponding TB595 FL- ORF sequence was introduced into the p2GW7 high copy vector (Karimi et al. 2002) in which the gene is put under the control of the p35S promoter, using the GatewayTM technology (InVitrogen Life Technologies).
  • promoter sequences of taxol synthesis genes from Taxus species synthetic DNA was made based by GenScript USA Inc for the promoters of taxadiene synthase (p7XS, 1436 nt, EMBL accession EF153471) and taxane 13-a-hydroxylase (pT130H, 1060 nt, EMBL accession EF153469).
  • Promoter sequences were directly synthesized between attB sites (GatewayTM technology; InVitrogen Life Technologies) and recombined into the GatewayTM entry vector pDONR221 and subsequently to the pGWL7 vector (Karimi et al. 2005), to create the reporter constructs in which the promoters are fused in front of the firefly luciferase open reading frame (pTXS-fLUC and pT130H-fLUC).
  • EXAM PLE 3 Functional analysis of the TB595 gene by transient expression in plant protoplasts with heterologous alkaloid synthesis reporter genes.
  • Promoter sequences for the Catharanthus roseus strictosidine synthase (pSTR; EM BL accession Y10182), and the Nicotiana tabacum putrescine N-methyltransferase (pPMTl; EM BL accession AF126810), and quinolinate phosphoribosyltransferase (pQPRT2; EM BL accession AJ748263) genes were PCR amplified between the GatewayTM attB sites, and GatewayTM recombined into the pDON R221 vector and subsequently to the pGWL7 vector (Karimi et al.
  • TB595 was also co-transfected with the pQPRT2 promoter and the known regulatory transcription factors (TFs) of nicotine biosynthesis in Nicotiana species, NtORCl (De Sutter et al. 2005) and NbbH LH l (Todd et al. 2010). It could be demonstrated that transfection with TB595 and MeJA has a synergistic effect on the combined activity of these two TFs in the presence of MeJA (Fig. 3).
  • EXAMPLE 4 Functional analysis of the TB595 gene by stable transformation of Taxus cells.
  • the corresponding TB595 FL-O F sequence was introduced in the pK7WGD2 binary vector (Karimi et al. 2002) in which the gene is put under the control of the p35S promoter, using the GatewayTM technology (InVitrogen Life Technologies).
  • the ternary vector system van der Fits et al. 2000 was applied.
  • the plasmid pBBRlMCS-5.virGN54D was used as a ternary vector.
  • the binary plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 already bearing the ternary plasmid by electro- transformation.
  • the A. tumefaciens PM P90-C58Cl nf strain was used.
  • the binary plasmid was introduced in the Agrobacterium rhizogenes strain LBA94.2/12.
  • Taxoid content is analysed by HPLC as previously described (Bonfill et al. 2007; Ketchum et al. 2007).
  • EXAMPLE 5 Functional analysis of the TB595 gene by stable transformation of other medicinal plant cells.
  • the ternary vector system van der Fits et al. 2000 was applied.
  • the plasmid pBBRlMCS-5.virGN54D was used as a ternary vector.
  • the binary plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 already bearing the ternary plasmid by electro- transformation.
  • the binary plasmid was introduced in the Agrobacterium tumefaciens strain PMP90-C58Clrif.
  • For hairy root transformation the binary plasmid was introduced in the Agrobacterium rhizogenes strain LBA94.2/12.
  • the resulting Agrobacterium strains were used to create transgenic plant (Nicotiana tabacum), hairy root (Nicotiana tabacum and Medicago truncatula) or cell lines (Nicotiana tabacum).
  • Leaves of Nicotiana tabacum cv. Xanthi plantlets grown in in vitro conditions were used for the establishment of hairy roots, after their infection with Agrobacterium rhizogenes LBA 9402 carrying the pK7WG2D expression vector with TB595 or GUS under the control of CaMV 35S promoter, and the nptll gene as a marker.
  • the Agrobacterium was cultured on solid YEB medium supplemented with 100 mg/L spectinomycin, 300 mg/L Streptomycin and lOOmg/L rifampicin in the dark at 28 Q C during 48h.
  • Leaves were infected by wounding the mid-ribs with a sterile needle inoculated with the desired Agrobacterium strain. Hairy roots appeared 2-4 weeks after the infection and they were excised and cultured individually on MS medium supplemented with 30 g/l sucrose and 500 mg/L cefotaxime to eliminate the bacteria and 100 mg/L kanamycin for hairy root selection. Hairy root lines were kept in the dark at 25 °C and routinely subcultured every 3-4 weeks.
  • Nicotine alkaloids were extracted modifying the protocol described in Sheng et al. (2005). 100 ⁇ 20 mg powdered lyophilized roots were resuspended in 200 ⁇ NH3-H20 6% and 4 mL methanohdichloromethane in 1:3 (v:v), kept in an ultrasound bath for 15 min and filtered. After evaporation, the supernatant was washed 3 times with methyl tert-butyl ethendichlormethane (MTBE:DCM) in 1:0.3 (v:v), evaporated again and kept at -20 Q C. Finally, just before its analysis, the extract was resuspended in 400 ⁇ MTBE:DCM 1:0.3 (v:v).
  • the samples were determined using a Flame Ionization Detector (FID) Gas Chromatography system (Hewlett-Packard HP5890 Series II Plus).
  • FID Flame Ionization Detector
  • the analyses were performed on an RTX-35 amine column (30 m, 0.25 mm i.d. x 0.5 ⁇ d.f.) by using split mode, with lmL/min of Helium flux and a programmed oven temperature from 60 Q C to 240 Q C, increasing 10 Q C/min. Injector and detector temperatures were kept at 270 Q C. Aliquots of 1 ⁇ were injected by a split-splitless 6890 AutoSampler (Agilent Technologies).
  • the nicotine alkaloids were identified by comparing retention times with the standards: Nicotine, Nornicotine (LGC Promochem, Barcelona, Spain), Anabasine (Chromadex, Irvine, CA, USA) and Anatabine (TransMIT, Marburg, Germany). Expression of the TB595 gene in transgenic tobacco roots was verified and confirmed with RT-PCR (Hakkinen et al., 2007).
  • the main alkaloid found both in control and TB595 root lines was nicotine. This alkaloid was produced in quantities 30-50 times higher than the other alkaloids studied.
  • the improvement of nicotine production in response to MeJA elicitation was significantly higher in the TB595 root lines (annotated with 1, 6, 7, 9, 10, 11, and 12 in Fig. 4) than in the control lines (annotated with CI, C2 and Cll in Fig. 4). Nicotine content in the TB595 root lines increased, on average, 3.5 times after MeJA elicitation, whereas the average increase observed in the control root lines was only 2.2 (Fig. 4). This analysis demonstrated that TB595 can stimulate nicotine biosynthesis in tobacco roots.
  • A. rhizogenes mediated transformation of M. truncatula (ecotype Jemalong J5) hairy roots was done according to a protocol adapted from Boisson-Dernier et al. (2001). Expression of the TB595 gene in transgenic Medicago roots was confirmed with RT-PCR. Medicago flavonoid and triterpene saponin content is analysed by Reversed phase UPLC/ICR/FT-MS (Pollier et al., 2011).
  • Metabolite extracts for saponin profiling are obtained from M. truncatula hairy roots grown for 21 days in liquid medium. The hairy roots are harvested and rinsed with purified water under vacuum filtration and snap-frozen in liquid nitrogen. Subsequently, the roots are ground in liquid nitrogen, and 400 mg of ground material is extracted with 1 ml methanol at room temperature for 10 minutes, followed by centrifugation for 10 minutes at 14000 rpm. 500 ⁇ of the supernatant is evaporated to dryness under vacuum and the residue is treated with 400 ⁇ water and 200 ⁇ cyclohexane. Samples are centrifugated (10 minutes at 14000 rpm) and 200 ⁇ of the aqueous phase is retained for analysis.
  • an Acquity UPLCTM BEH C18 column (150 x 2.1 mm, 1.7 ⁇ ; Waters, Milford, MA) is serially coupled to an Acquity UPLCTM BEH C18 column (100 x 2.1 mm, 1.7 ⁇ ) and mounted on an ultrahigh performance LC system consisting of a Accela pump (Thermo Electron Corporation, Bremen, Germany) and Accela autosampler (Thermo Electron Corporation).
  • the Accela LC system is hyphenated to a LTQ FT Ultra (Thermo Electron Corporation) via an electrospray ionization source.
  • Full FT-MS spectra between m/z 120-1400 are recorded at a resolution of 100,000.
  • full MS spectra are interchanged with a dependent MS 2 scan event in which the most abundant ion in the previous full MS scan are fragmented, and two dependent MS 3 scan events of the two most abundant daughter ions.
  • the collision energy is set at 35%.
  • EXAMPLE 6 Functional analysis of TB595 orthologs from other plant species.
  • the phylogenetic tree was generated in MEGA 4.0.1 software (Tamura et al., 2007), by the Neighbor-Joining method, and bootstrapping was done with 10,000 replicates.
  • the evolutionary distances were computed with the Poisson correction method, and all positions containing gaps and missing data were eliminated from the data set (complete deletion option).
  • This analysis indicated that TB595 is most related to A. thaliana At2g20562 and M. truncatula MtTC163920 (Fig. 6). Besides these genes, A. thaliana and M. truncatula possess 1 and 7 additional isoforms of the TB595 gene, respectively (Fig. 6).
  • the binary plasmid was introduced in the Agrobacterium rhizogenes strain LBA94.2/12.
  • the resulting Agrobacterium strains were used to create transgenic plant (Arabidopsis thaliana), or hairy root (Medicago truncatula) lines.
  • A. rhizogenes mediated transformation of M. truncatula (ecotype Jemalong J5) hairy roots with the TB595-like genes was done according to a protocol adapted from Boisson-Dernier et al. (2001). Expression of the TB595 orthologs in transgenic Medicago roots was verified and confirmed with RT- PCR. Medicago flavonoid and triterpene saponin content is analysed by Reversed phase UPLC/ICR/FT- MS (Pollier et al., 2011), as further detailed in Example 5.
  • Extracts are analyzed with an Accela ultrahigh performance liquid chromatography (UHPLC) system (Thermo Electron Corporation, Bremen, Germany) consisting of an Accela autosampler coupled to an Accela pump and further hyphenated to a LTQ FT Ultra (Thermo Electron Corporation) mass spectrometer (MS) consisting of a linear ion trap (IT) MS connected with a Fourier transform ion cyclotron resonance (FT-ICR) MS.
  • UHPLC Accela ultrahigh performance liquid chromatography
  • MS mass spectrometer
  • the separation is performed on a reversed phase Acquity UPLC HSS C18 column (150 mm x 2.1 mm, 1.8 ⁇ ; Waters, Milford, MA) with aqueous 0.1% acetic acid and acetonitrile/water (99/1, v/v, acidified with 0.1% acetic acid) as solvents A and B.
  • aqueous 0.1% acetic acid and acetonitrile/water 99/1, v/v, acidified with 0.1% acetic acid
  • the following gradient is applied: 0 min 1% B, 30 min 60% B, 35 min 100% B.
  • the autosampler temperature is 5°C.
  • Analytes are negatively ionized with an electrospray ionization (ESI) source using the following parameter values: spray voltage 3.5 kV, capillary temperature 300°C, sheath gas 40 (arb), aux gas 20 (arb).
  • ESI electrospray ionization
  • Full FT-ICR-MS spectra between 120 and 1400 m/z are recorded (1.2-1.7 sec/scan) at a resolution of 100,000.
  • MSn scans are obtained with 35% collision energy.
  • the TB595 O F encodes for a peptide of 73 amino acids, of which the first 27 aa putatively correspond to signal peptide, as predicted by the SignalP software (Fig. 7 & 8) (Emanuelsson et al. 2007).
  • the predicted mature peptide consists of 46 aa, and its sequence was highly conserved in the plant kingdom, as determined with the ClustalW software (Fig. 5 & 8), see also Example 6 (Larkin et al. 2007).
  • the ORF encoding the marker protein Venus-GFP was fused to the C-terminus of TB595, and the fusion gene put under the control of the p35S promoter in a binary vector, using the GatewayTM technology (InVitrogen Life Technologies).
  • the binary plasmid was introduced into the Agrobacterium tumefaciens strain and the resulting strain used to perform either leaf infiltrations of Nicotiana benthamiana plants as described in Boruc et al. (2010) or stable transformation of Nicotiana tabacum BY-2 cells and Arabidopsis thaliana cells as described (Goossens et al. 2003; Pauwels et al., 2010).
  • HIS or FLAG tagged TB595 peptide for expression and purification from E. coli is generated.
  • HIS or FLAG tag cleavage recognition sites incorporated between the tag and the peptide itself can be used to obtain the native peptide post-purification (http://www.qiagen.com/products/protein/purification/tagzymesystem/default.aspx).
  • the peptide Due to the highly hydrophobic nature of the peptide the peptide is also expressed with an excretory signal in yeast. This is achieved by generating a fusion of the M Falpha-1 prepro-leader sequence (89aa; Rothblatt et al. 1987) N-terminal to the HIS-tagged TB595 for excretion of the tagged protein into the medium. The peptide is subsequently purified using the HIS tag before it is cleaved to obtain the native peptide.
  • the pure synthetic or recombinant peptides are applied in various concentrations to suspension cells,hairy roots and whole plants from various medicinal plant species, in particular Taxus baccata, Nicotiana tabacum, and Medicago truncatula, in which we subsequently analyse accumulation of taxoids, pyridine alkaloids and triterpene saponins/flavonoids, respectively.
  • Floral dip a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
  • MEGA4 Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596 1599.

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Abstract

La présente invention concerne des polypeptides isolés et des homologues de ceux-ci, et leur utilisation pour l'amélioration de la production de métabolites secondaires dans des plantes ou des cellules végétales. L'invention concerne également des polynucléotides codant de tels polypeptides et des gènes chimères, ainsi que des vecteurs, des cellules hôtes et des plantes transgéniques les contenant. L'invention concerne également des procédés pour la production ou la stimulation de la biosynthèse de métabolites secondaires dans des végétaux ou des cellules végétales.
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US11485981B2 (en) 2013-03-14 2022-11-01 Monsanto Technology Llc Plant regulatory elements derived from medicago truncatula 3′UTR sequences, and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
WO2007072224A2 (fr) * 2006-09-13 2007-06-28 Nara Institute Of Science And Technology Augmentation des niveaux d'alcaloides nicotiniques
US20080292735A1 (en) * 2006-11-17 2008-11-27 Nara Institute Of Science And Technology Regulating alkaloids
WO2009063312A2 (fr) * 2007-05-25 2009-05-22 National Research Council Of Canada Séquences d'acides nucléiques codant les facteurs de transcription qui régulent la biosynthèse alcaloïdique et leur utilisation dans la modification du métabolisme des plantes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
WO2007072224A2 (fr) * 2006-09-13 2007-06-28 Nara Institute Of Science And Technology Augmentation des niveaux d'alcaloides nicotiniques
US20080292735A1 (en) * 2006-11-17 2008-11-27 Nara Institute Of Science And Technology Regulating alkaloids
WO2009063312A2 (fr) * 2007-05-25 2009-05-22 National Research Council Of Canada Séquences d'acides nucléiques codant les facteurs de transcription qui régulent la biosynthèse alcaloïdique et leur utilisation dans la modification du métabolisme des plantes

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
"Plant Cell Culture", 1985, IRL PRESS, LTD.
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403
ALTSCHUL, S.F.; MADDEN, T.L.; SCHAFFER, A.A.; ZHANG, J.; ZHANG, Z.; MILLER, W.; LIPMAN, D.J.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1992, GREENE PUBLISHING ASSOCIATES
BARCELO, P.; LAZZERI, P.A.: "Transgenic Plant Research", 1998, HARWOOD ACADEMIC PUBLISHERS, article "Direct gene transfer: chemical, electrical and physical methods", pages: 35 - 55
BOISSON-DERNIER, A.; CHABAUD, M.; GARCIA, F.; BECARD, G.; ROSENBERG, C.; BARKER, D.G.: "Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen- fixing and endomycorrhizal symbiotic associations", MOL. PLANT MIC. INT., vol. 14, 2001, pages 695 - 700
BONFILL, M.; BENTEBIBEL, S.; MOYANO, E.; PALAZ6N, J.; CUSIDO, R.M.; EIBL, R.; PIFIOL, M.T.: "Paclitaxel and baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata", BIOL. PLANT., vol. 51, 2007, pages 647 - 652, XP019548840, DOI: doi:10.1007/s10535-007-0137-2
BORUC, J.; VAN DEN DAELE, H.; HOLLUNDER, J.; ROMBAUTS, S.; MYLLE, E.; HILSON, P.; NZE, D.; DE VEYLDER, L.; RUSSINOVA, E.: "Functional modules in the Arabidopsis core cell cycle binary protein-protein interaction network", PLANT CELL, vol. 22, 2010, pages 1264 - 1280
CHICO, J.M.; CHINI, A.; FONSECA, S.; SOLANO, R.: "JAZ repressors set the rhythm in jasmonate signaling", CURR. OPIN. PLANT BIOL., vol. 11, 2008, pages 486 - 494, XP025431810, DOI: doi:10.1016/j.pbi.2008.06.003
CLOUGH, S. J.; BENT, A. F.: "Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana", PLANT J., vol. 16, 1998, pages 735 - 743, XP002132452, DOI: doi:10.1046/j.1365-313x.1998.00343.x
CROTEAU, R.; KETCHUM, R.E.; LONG, R.M.; KASPERA, R.; WILDUNG, M.R.: "Taxol biosynthesis and molecular genetics", PHYTOCHEM REV., vol. 5, 2006, pages 75 - 97, XP019405391, DOI: doi:10.1007/s11101-005-3748-2
DATABASE Tair [Online] 29 September 2008 (2008-09-29), XP002679284, retrieved from arabidopsis Database accession no. AT2G20562 *
DE SUTTER, V.; VANDERHAEGHEN, R.; TILLEMAN, S.; LAMMERTYN, F.; VANHOUTTE, I.; KARIMI, M.; NZE, D.; GOOSSENS, A.; HILSON, P.: "Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells", PLANT J., vol. 44, 2005, pages 1065 - 1076
DURZAN; VENTIMIGLIA, IN VITRO CELL DEV. BIOL., vol. 30, 1994, pages 219 - 227
EMANUELSSON, O.; BRUNAK, S.; VON HEIJNE, G.; NIELSEN, H.: "Locating proteins in the cell using TargetP, SignalP, and related tools", NAT. PROTOC., vol. 2, 2007, pages 953 - 971, XP008097990, DOI: doi:10.1038/nprot.2007.131
EXPOSITO, O.; SYKLOWSKA-BARANEK, K.; MOYANO, E.; ONRUBIA, M.; BONFILL, M.; PALAZON, J.; CUSIDO R.M.: "Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate", BIOTECHNOL. PROG., vol. 26, 2010, pages 1145 - 1153
FLORES, H. E.; PROTACIO, C.M.; SIGNS, M.W.: "Plant nitrogen metabolism", RECENT ADV. PHYTOCHEM., vol. 23, 1991, pages 329
G. SAMUELSSON, DRUGS OF NATURAL ORIGIN, 1999
GEORGE ET AL.: "Plant Culture Media", vol. 1, 1987, FORMULATIONS AND USES EXEGETICS LTD.
GHEYSEN, G.; ANGENON, G.; VAN MONTAGU, M.: "Transgenic Plant Research", 1998, HARWOOD ACADEMIC PUBLISHERS, article "Agrobacterium-mediated plant transformation: a scientifically intriguing story with significant applications", pages: 1 - 33
GOLDMANN ET AL., PHYTOCHEMISTRY, vol. 29, 1990, pages 2125
GOOSSENS, A.; HAKKINEN, S.T.; LAAKSO, I.; OKSMAN-CALDENTEY, K.M.; INZÉ D.: "Secretion of secondary metabolites by ATP-binding cassette transporters in plant cell suspension cultures", PLANT PHYSIOL., vol. 131, 2003, pages 1161 - 1164
HAKKINEN, S.T.; TILLEMAN, S.; SWIATEK, A.; DE SUTTER, V.; RISCHER, H.; VANHOUTTE, I.; VAN ONCKELEN, H.; HILSON, P.; NZE, D.; OKSMA: "Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco", PHYTOCHEMISTRY, vol. 68, 2007, pages 2773 - 2785
HERMAN E.B., REGENERATION AND MICROPROPAGATION: TECHNIQUES, SYSTEMS AND MEDIA 1997-1999, 2000
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151
KARIMI, M'' NZE, D.; DEPICKER, A.: "GATEWAY vectors for Agrobacterium-mediated plant transformation", TRENDS PLANT SCI., vol. 7, 2002, pages 193 - 195, XP002714178, DOI: doi:10.1016/S1360-1385(02)02251-3
KARIMI, M.; DE MEYER, B.; HILSON, P.: "Modular cloning and expression of tagged fluorescent protein in plant cells", TRENDS PLANT SCI., vol. 10, 2005, pages 103 - 105
KETCHUM, R.E.; WHERLAND, L.; CROTEAU, R.B.: "Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures", PLANT CELL REP., vol. 26, 2007, pages 1025 - 1033, XP019539982, DOI: doi:10.1007/s00299-007-0323-x
LARKIN, M.A.; BLACKSHIELDS, G.; BROWN, N.P.; CHENNA, R.; MCGETTIGAN, P.A.; MCWILLIAM, H.; VALENTIN, F.; WALLACE, I.M.; WILM, A.; L: "Clustal W and Clustal X version 2.0", BIOINFORMATICS, vol. 23, 2007, pages 2947 - 2948
MILLET, A.; STINTZING, F.; MERFORT, 1.: "Validation of GC-FID method for rapid quantification of nicotine in fermented extracts from Nicotiana tabacum fresh leaves and studies of nicotine metabolites", J. PHARM. BIOMED. ANAL., vol. 49, 2009, pages 1166 - 1171, XP026089959, DOI: doi:10.1016/j.jpba.2009.02.024
PAUWELS, L., NZE, D.; GOOSSENS, A.: "Jasmonate-inducible gene: what does it mean?", TRENDS PLANT SCI., vol. 14, 2009, pages 87 - 91, XP025916734, DOI: doi:10.1016/j.tplants.2008.11.005
PAUWELS, L.; BARBERO, G.F.; GEERINCK, J.; TILLEMAN, S.; GRUNEWALD, W.; PEREZ, A.C.; CHICO, J.M.; BOSSCHE, R.V.; SEWELL, J.; GIL, E: "NINJA connects the co-repressor TOPLESS to jasmonate signalling", NATURE, vol. 464, 2010, pages 788 - 791
POLLIER, J.; MORREEL, K.; GEELEN, D.; GOOSSENS, A.: "Metabolite profiling of triterpene saponins in Medicago truncatula hairy roots by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry", J. NAT. PROD., vol. 74, 2011, pages 1462 - 1476
RISCHER, H.; ORESIC, M.; SEPPANEN-LAAKSO, T.; KATAJAMAA, M.; LAMMERTYN, F.; ARDILES-DIAZ, W.; VAN MONTAGU, M.C.E.; NZE, D.; OKSMAN: "Gene to metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells", PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 5614 - 5619
ROTHBLATT, J.A.; WEBB, J.R.; AMMERER, G.; MEYER, D.I.: "Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro", EMBO J., vol. 6, 1987, pages 3455 - 3463, XP001316729
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SHENG, L.Q.; DING, L.; TONG, H.W.; YONG, G.P.; ZHOU, X.Z.; LIU, S.M.: "Determination of nicotine- related alkaloids in tobacco and cigarette smoke by GC-FID", CHROMATOGRAPHIC, vol. 62, 2005, pages 63 - 68, XP019358442
STAFFORD, H.A., BOTANICAL REVIEW, vol. 66, 2000, pages 99 - 118
SUZUKI, K.; YUN, D.J.; CHEN, X.-Y.; YAMADA, Y.; HASHIMOTO, T.: "An Atropa belladonna hyoscyamine 6(3-hydro!ase gene is differentially expressed in the root pericycle and anthers", PLANT MOL. BIOL., vol. 40, pages 141
TAMURA, K.; DUDLEY, J.; NEI, M.; KUMAR, S.: "MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0", MOL. BIOL. EVOL., vol. 24, 2007, pages 1596 - 1599
TODD, A.T.; LIU, E.; POLVI, S.L.; PAMMETT, R.T.; PAGE, J.E.: "A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana", PLANT J., vol. 62, 2010, pages 589 - 600
TOIVOUNEN L., BIOTECHNOL. PROG., vol. 9, 1993, pages 12
TRIGIANO R.N.; GRAY D.J., PLANT TISSUE CULTURE CONCEPTS AND LABORATORY EXERCISES, 1999
TURPEN TH, PHILOS TRANS R SOC LOND B BIOL SCI, vol. 354, no. 1383, 1999, pages 665 - 73
VAN DER FITS, L.; DEAKIN, E.A.; HOGE, J.H.; MEMELINK J.: "The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation", PLANT MOL. BIOL., vol. 43, 2000, pages 495 - 502, XP003020231, DOI: doi:10.1023/A:1006440221718
VANHALA L. ET AL., PLANT CELL REP., vol. 14, 1995, pages 236
VERPOORTE R; VAN DER HEIJDEN R; MEMELINK J.: "Engineering the plant cell factory for secondary metabolite production", TRANSGENIC RES., vol. 9, 2000, pages 323 - 43, XP009030290, DOI: doi:10.1023/A:1008966404981
VERPOORTE, R. ET AL., BIOTECHNOL. LETT., vol. 21, 1999, pages 467
VUYLSTEKE, M.; PELEMAN, J.D.; VAN EIJK, M.J.: "AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis", NAT. PROTOC., vol. 2, 2007, pages 1399 - 1413, XP008120512, DOI: doi:10.1038/nprot.2007.174
ZHAO, J.; DAVIS, L.C.; VERPOORTE R.: "Elicitor signal transduction leading to production of plant secondary metabolites", BIOTECHNOL. ADV., vol. 23, 2005, pages 283 - 333, XP004852805, DOI: doi:10.1016/j.biotechadv.2005.01.003

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US11485981B2 (en) 2013-03-14 2022-11-01 Monsanto Technology Llc Plant regulatory elements derived from medicago truncatula 3′UTR sequences, and uses thereof

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