WO1997015584A2 - Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs - Google Patents

Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs Download PDF

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WO1997015584A2
WO1997015584A2 PCT/US1996/016482 US9616482W WO9715584A2 WO 1997015584 A2 WO1997015584 A2 WO 1997015584A2 US 9616482 W US9616482 W US 9616482W WO 9715584 A2 WO9715584 A2 WO 9715584A2
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nucleic acid
sequence
acid segment
linalool
seq
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PCT/US1996/016482
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WO1997015584A3 (fr
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Eran Pichersky
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The Regents Of The University Of Michigan
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Priority to CA 2234647 priority Critical patent/CA2234647A1/fr
Priority to EP96939462A priority patent/EP0858507A2/fr
Priority to AU76632/96A priority patent/AU7663296A/en
Publication of WO1997015584A2 publication Critical patent/WO1997015584A2/fr
Publication of WO1997015584A3 publication Critical patent/WO1997015584A3/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • 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

  • the present invention relates generally to the fields of the floral emission of monoterpenes and the production of monoterpenes by plants.
  • the present invention also relates to the field of the production of enhanced scent and taste in plants.
  • the scent emitted by such flowers is often a complex mixture of low molecular weight compounds, and the relative abundances and interactions of the constituents give the flower its particular characteristic fragrance.
  • Floral scents have been demonstrated to function as long and short-distance attractants and nectar guides to a variety of animal pollinators (reviewed in Dobson, 1993).
  • insects are able to distinguish between complex floral scent mixtures (Dodson et al., 1969; Pellmyr, 1986).
  • Clarkia breweri an annual plant native to California, has a strong, sweet fragrance, consisting of some 8-12 different volatiles that fall into two groups: monoterpenoids and benzenoids (Raguso and
  • linalool an acyclic monoterpene common to the floral scents of numerous other plant species (Kaiser, 1991; Knudsen et al., 1993).
  • Two cyclic isomers of linalool oxide are also produced by C. breweri (FIG. 1), almost certainly by further oxidative modification of linalool.
  • Monoterpenes are a large and diverse group of natural products. Due to their volatility, and thus their ability to be perceived at a distance, they are often involved in plant-insect interactions (Harborne, 1991; Langenheim, 1994). In addition to pollinator attraction (Dobson et al., 1993; Knudsen et al., 1993b), monoterpenes also play an important role in plant defense (Langenheim, 1994; Lewinsohn et al., 1992a) or may act as semio-chemicals (Turlings et al., 1990; Gijzen, 1993). Monoterpenes are also of commercial value as essential oils for perfumery and flavoring use and as industrial raw materials (Dawson, 1994).
  • Monoterpenes are derived from the ubiquitous isoprenoid intermediate GPP by a class of enzymes called monoterpene synthases (also termed cyclases when they catalyze the formation of cyclic products). Although many monoterpene synthases from plants have been described, only a few of these enzymes have been purified to homogeneity and
  • the present invention seeks to overcome these and other drawbacks inherent in the prior art by providing a purified linalool synthase polypeptide and by further providing an isolated nucleic acid segment encoding the linalool synthase polypeptide.
  • an important embodiment of the invention may be described as an isolated nucleic acid segment comprising a nucleic acid sequence encoding a linalool synthase protein or polypeptide.
  • the nucleic acid sequence may encode an S-linalool synthase polypeptide and may more particularly encode a Clarkia breweri S-linalool synthase polypeptide.
  • the isolated nucleic acid segment of the present invention may encode the amino acid sequence disclosed herein as SEQ ID NO: 2.
  • the invention may also be described as a nucleic acid segment comprising a nucleic acid sequence consisting essentially of the nucleic acid sequence of SEQ ID NO:1. It is evident, due to the degeneracy of the genetic code and the functional equivalency of certain amino acids within a polypeptide sequence, that nucleic acid sequences may vary
  • nucleic acid sequence may be altered for various reasons that include, but are not limited to the use of codons that occur more frequently in the gene sequences of a particular host organism, or to insert or delete a restriction enzyme recognition site for ease in moving the particular sequence into or out of a vector, without altering the function of the
  • embodiment of the present invention is a nucleic acid segment that has the nucleic acid sequence of SEQ ID NO:1, certain variations as described are also
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above.
  • nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3 ' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleic acid segment is intended to refer to a nucleic acid molecule which has been isolated free of total cellular DNA or RNA, as the case may be, of a particular species. Therefore, a nucleic acid segment encoding a linalool synthase is intended to refer to a nucleic acid segment which
  • nucleic acid segment contains such coding sequences yet is isolated away from total RNA or DNA of a Clarkia breweri cell. Included within the term “nucleic acid segment”, are DNA segments, whether isolated from genomic or cDNA sources or even prepared synthetically, and RNA segments which may be isolated mRNA or RNA obtained by in vi tro or in vivo transcription of a DNA segment, or a chemically
  • RNA molecules synthesized RNA molecule.
  • the term also includes DNA or RNA segments which may be employed in the preparation of vectors, as well as the vectors themselves, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • DNA composition may be used for delivery to recipient cells in accordance with the present invention.
  • Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the Lis gene.
  • DNA constructs can further include structures such as promoters, enhancers,
  • polylinkers or even regulatory genes as desired.
  • the nucleic acid segments of the present invention may be positioned under the control of a promoter, and may even be positioned under the control of a recombinant promoter.
  • the promoter may be in the form of the promoter which is naturally associated with a linalool synthase gene in Clarkia breweri cells, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a linalool synthase gene in its natural environment.
  • Preferred constructs will generally include a plant promoter such as the CaMV 35S promoter (Odell et al., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang & Russell, 1990),
  • Tissue specific promoters such as root cell promoters (Conkling et al., 1990) and tissue specific enhancers (Fromm et al., 1989) are also known.
  • inducible promoters such as ABA- and turgor-inducible promoters.
  • promoter that effectively directs the expression of the nucleic acid segment in the cell type chosen for expression.
  • the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, (for example, see Sambrook et al . (1989); Methods in Plant Molecular Biology and
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced nucleic acid segment, such as is
  • promoter systems contemplated for use in high-level expression include, but are not limited to, the T7 RNA polymerase promoter system described by Tabor & Richardson (1985) and the maltose binding protein-fusion protein system (Nagai & Thogersen, 1987). Constructs will also include the Lis gene along with a 3 ' end DNA sequence that acts as a signal to terminate transcription and allow for the poly-adenylation of the resultant mRNA.
  • the most preferred 3' elements are contemplated to be those from the nopaline synthase gene of Agrobacterium tumefasciens (Bevan et al., 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefasciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.
  • Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989), may further be included where desired.
  • leader sequences are DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence.
  • Preferred leader sequences are
  • sequences predicted to direct optimum expression of the attached gene i.e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation.
  • sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants, and in floral or fruit tissue in particular, will be most preferred. It is contemplated that vectors for use in
  • oca enhancer element may be constructed to include the oca enhancer element.
  • This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of agrobacterium (Ellis et al., 1987), and is present in at least 10 other promoters (Bouchez et al., 1989). It is proposed that the use of an enhancer element, such as the oca element and
  • promoters when applied in the context of transformation.
  • tissue-specific promoter sequences for use in accordance with the present invention. To achieve this, one may first isolate cDNA clones from the tissue concerned and
  • the production of a transformed cell includes the introduction of an exogenous DNA segment, such as a cDNA or gene, into a recipient cell to create the transformed cell.
  • an exogenous DNA segment such as a cDNA or gene
  • the frequency of occurrence of cells receiving DNA is believed to be low.
  • it is most likely that not all recipient cells receiving DNA segments will result in a transformed cell wherein the DNA is stably integrated into the plant genome and/or expressed. Some may show only initial and transient gene expression.
  • Suitable methods are believed to include virtually any method by which DNA can be introduced into a plant cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc.
  • acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes, may be employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells.
  • recipient cells are made more susceptible to transformation, by mechanical wounding.
  • friable tissues such as a suspension culture of cells, or embryogenic callus, or
  • pectolyases pectolyases
  • particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming plant cells, is that neither the isolation of protoplasts (Cristou et al., 1988) nor the susceptibility to Agrobacterium infection is required.
  • An illustrative embodiment of a method for delivering DNA into cells by acceleration is a Biolistics Particle
  • Delivery System which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with cells cultured in suspension.
  • the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile
  • apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • the number of cells in a focus which express the exogenous gene product 48 hours postbombardment often range from 1 to 10 and average 1 to 3.
  • bombardment transformation one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable
  • Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro-or microprojectiles.
  • Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.
  • TRFs trauma reduction factors
  • nucleic acid segments of the present invention may also be defined as recombinant vectors when the nucleic acid sequences disclosed and described herein are combined with, or joined to nucleic acid sequences that allow the transformation of those sequences into a host cell, and in some cases, replication of those sequences in the host cell.
  • the nucleic acid segments may be defined as a recombinant expression vector capable of expressing a linalool synthase protein or polypeptide on introduction into a host cell, or alternatively as a plant transformed with such a recombinant expression vector.
  • the vector comprises a nucleic acid sequence in accordance with SEQ ID NO:1, and the vector may further comprise the pBLUESCRIPT or pBIN19 nucleic acid sequence.
  • the present invention may, in certain embodiments be defined as a recombinant host cell comprising a nucleic acid segment that encodes a linalool synthase
  • the recombinant host cell may be a
  • prokaryotic cell such as a bacterial cell, or E. coli cell
  • the recombinant host cell may be a eukaryotic cell, with a preferred eukaryotic cell being a yeast cell or a plant cell.
  • the plant cell may be a part of a plant or a substructure of a plant. It is understood that the nucleic acid segment contained in the host cell may be positioned under the control of a promoter and further that the nucleic acid segment may be positioned in a recombinant vector.
  • the recombinant vector may also be a recombinant expression vector wherein the host cell expresses a linalool synthase polypeptide.
  • the invention may be any organic compound. In certain embodiments, the invention may be any organic compound.
  • nucleic acid segment hybridizable to a nucleic acid segment comprising the sequence of SEQ ID NO:1 under stringent conditions, or even as consisting essentially of the complement of SEQ ID NO:1.
  • Stringent conditions are defined as relatively low salt and ⁇ or high temperature, such as provided by 0.02M-0.15M NaCl or the equivalent at temperatures of 50°C to 70°C.
  • the term "complement" is used to define the strand of nucleic acid which will hybridize to the first nucleic acid sequence to form a double stranded molecule under stringent conditions.
  • hybridization at low temperature and/or high ionic strength is termed low stringency and hybridization at high temperature and/or low ionic strength is termed high stringency.
  • the temperature and ionic strength of a desired stringency are understood to be applicable to particular probe lengths, to the length and base content of the sequences and to the presence of formamide in the hybridization mixture. It is understood in the art that a nucleic acid sequence will hybridize with a complementary nucleic acid sequence under high stringency conditions even though some mismatches may be present. Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present claimed invention.
  • nucleic acid sequences disclosed herein will also find utility as probes or primers in nucleic acid hybridization embodiments.
  • oligonucleotides which comprise a sequence of at least 15, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1500 or even 2000 contiguous nucleotides which corresponds to at least a 15, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1500 or even 2000 nucleotide contiguous sequence of SEQ ID NO:1 or its complement will be useful as probes or primers.
  • nucleic acid segments comprising a sequence of at least 2,583 contiguous nucleotides which corresponds to a sequence of at least 2,583 contiguous nucleotides of SEQ ID NO:1 or its complement, such as the 2,583 contiguous nucleotides of SEQ ID NO:1 that encode the mature protein, for example will be useful for various embodiments, as will a segment comprising a sequence of at least 2,610
  • contiguous nucleotides which corresponds to a sequence of at least 2,610 contiguous nucleotides of SEQ ID NO:1 or its complement, such as the 2,610 nucleotide sequence encoding the entire coding region of SEQ ID NO:1, or even a nucleic acid segment comprising a sequence of at least 2,681 contiguous nucleotides which corresponds to the 2,681 nucleotide sequence of SEQ ID NO:1 or its
  • nucleic acid segments may be contained in various vectors and described above, and that as such, a nucleic acid segment of the present invention may be further defined as a nucleic acid fragment derived from SEQ ID NO:1 or its complement comprising up to 10,000, 5,000, 3,000, 1,000, 500, 100 or even 50 basepairs in length.
  • the invention may also be described as a method of using a nucleic acid segment encoding a linalool synthase protein or polypeptide, comprising the steps of preparing a recombinant vector in which said nucleic acid segment is positioned under the control of a promoter; introducing said recombinant vector into a host cell; culturing said host cell under conditions effective to allow expression of the encoded linalool synthase protein or polypeptide; and collecting said expressed linalool synthase protein or polypeptide.
  • the invention may also be described in certain embodiments as a method of enhancing the scent production of a plant, comprising the steps of obtaining a
  • the nucleic acid segment may be under the control of a tissue specific
  • promoter/enhancer may preferably be specific for floral tissue.
  • the plant is a flowering plant, petunia, rose, carnation, etc., for example.
  • the present invention may also be described, in certain embodiments as a method of enhancing the flavor of a plant, comprising the steps of obtaining a
  • recombinant vector capable of expressing the nucleic acid segment encoding a linalool synthase polypeptide on introduction into a plant cell; transforming said plant with said vector; and growing said plant under conditions appropriate for expression of said nucleic acid segment, and preferably wherein the nucleic acia segment is under the control of a tissue specific promoter/enhancer, and more preferably wherein the promoter/enhancer is specific for fruit or leaf tissue.
  • tissue specific promoter/enhancer examples include, but are not limited to tomato, grape and tea plants.
  • An important embodiment of the present invention is a purified linalool synthase polypeptide having a molecular weight of from about 68 to 79 kDa and a specific activity of at least about 20 pkat/mg or even a specific activity of at least about 44.1 pkat/mg, or even more preferably a specific activity of at least about 395 pkat/mg. It is understood that a 1 pkat unit is defined as 1 picomole of product formed/second.
  • polypeptide composition of the present invention may be obtained by separating proteins from a flower or a flower part of a Clarkia breweri plant.
  • a method of obtaining a linalool synthase protein composition one would first obtain a protein extract by mechanical or enzymatic cellular disruption and centrifugation.
  • the further purification of such a desired protein is well known in the art and within the skill of the skilled practitioner.
  • Those fractions may be identified by measuring the absorbance at 280 nm, for example (to determine the presence of polypeptides), and assaying those fractions containing proteins for linalool synthase activity.
  • Those fractions containing the desired activity may again be further purified if so desired, by pooling those fractions and passing them over a hydroxyapatite column, for example, and again collecting fractions as before.
  • the collected fractions may be even further purified if desired, by passing those fractions over a Mono-Q column and collecting the fractions with linalool synthase activity to obtain said polypeptide.
  • the polypeptide obtained by this method may in certain preferred
  • a certain embodiment of the present invention is also a recombinant polypeptide comprising as a part of its amino acid sequence, a sequence according to the amino acid sequence set forth as SEQ ID NO: 2, or even a recombinant polypeptide that has the amino acid sequence of SEQ ID NO: 2.
  • a certain embodiment of the invention is also an antibody immunoreactive with the polypeptides as defined immediately above.
  • the antibody may be a polyclonal antibody, or is more preferably a monoclonal antibody.
  • GPP Geranyl Pyrophosphate
  • LIS Linalool Synthase
  • LSC Liquid Scintillation
  • DTT dithiothreitol
  • GC Gas chromatography
  • LPP Linalyl pyrophosphate
  • LIS S-Linalool synthase
  • Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • Tris tris(hydroxymethyl)-aminomethane.
  • FIG. 1 The formation of S-linalool from GPP by the action of S-linalool synthase and subsequent conversion to S-linalool oxides. S-Linalool synthase and
  • monoterpene cyclases have a similar ionization first step, leading to the intermediate linalyl cation (1).
  • S-linalool is formed by water addition in the reaction catalyzed by S-linalool synthase, whereas the bound LPP (either S- or R-LPP, depending on the particular cyclase) is isomerized and cyclized to cyclohexanoid monoterpenes by monoterpene cyclases.
  • FIG. 2 SDS-PAGE analysis of S-linalool synthase
  • M molecular weight markers (numbers at left indicate kDa). 1. Sample after DEAE-cellulose chromatography (16 ⁇ g protein); 2. Sample after hydroxyapatite chromatography (8 ⁇ g protein); 3. After Mono Q anion exchange FPLC (1 ⁇ g protein); 4. Same as in lane 3 but 2 ⁇ g protein. The gel was Coomassie-stained as described in Andrews, 1986.
  • FIG. 3 Gel permeation chromatography of the C. breweri S-linalool synthase.
  • the Mono Q purified enzyme was separated on a Superdex 75 (Pharmacia FPLC), and gave a molecular mass corresponding to 73 ⁇ 5 kDa by comparison with protein standards : yeast alcohol dehydrogenase (M r 150,000), bovine serum albumin (M r 67,000), hen egg white ovalbumin (M r 45,000), bovine carbonic anhydrase (M r 29,000) and lysozyme (M r 14,500).
  • yeast alcohol dehydrogenase M r 150,000
  • bovine serum albumin M r 67,000
  • hen egg white ovalbumin M r 45,000
  • bovine carbonic anhydrase M r 29,000
  • lysozyme M r 14,500
  • FIG. 4 Capillary radio-GC analysis of the product of S-linalool synthase.
  • the tracing in A is the radioactivity response to the pentane-soluble products generated by incubating C. breweri S-linalool synthase preparation with [ 3 H]-GPP.
  • the tracing in B is the thermal conductivity detector response to authentic geraniol and linalool standards.
  • C. breweri flowers A major component of the scent of C. breweri flowers is linalool, an acyclic monoterpene alcohol common to the floral scents of numerous other plant species (Knudsen et al., 1993a; Kaiser, 1991).
  • C. breweri flowers also synthesize and emit two linalool oxides, for which linalool is the proposed precursor (Winterhalter et al., 1986; Pichersky et al., 1994).
  • the present inventor has previously observed S-linalool synthase (LIS) activity in Clarkia flower parts
  • LIS is both developmentally and differentially regulated in the various floral organs (Pichersky et al., 1994). Total LIS activity per flower was highest in petals, from which most of the linalool emission occurs. LIS activity per fresh weight was highest in stigma and style (i.e., the pistil), but most of the linalool produced by these tissues is converted to linalool oxides by as yet unidentified enzymes.
  • the inventors report the purification and characterization of the S-linalool synthase from stigmata of C. breweri and the isolation and characterization of the cDNA gene encoding the S-linalool synthase.
  • concinna nor from other Clarkia species.
  • headspace analyses were repeated with 20-30 living C. concinna flowers combined, the compounds linalool, pyranoid linalool oxide and furanoid linalool oxide were detected, but at levels 1000-fold less than in C.
  • headspace To determine the amount of monoterpenes emitted at different stages of floral development, headspace
  • Determination of the level of the monoterpenes found in the flower and bud tissue indicates that there is a substantial pool of these compounds in the tissue. For example, at the time of peak emission (D2), the amount of linalool in the tissue is approximately equal to 10% of the total emitted over the corresponding 12 h period. Similar results were found for the linalool oxides. The observations that large pools of these monoterpenes exist within the tissues and that the changes in intracellular monoterpenes concentration parallel those of monoterpene emission (as does enzyme activity), suggest that these compounds are not sequestered for long as free
  • the Lis gene is not unique to C. breweri , but is found, and is active, in C. concinna . This finding raises the question of the function of the pathway in non-scented plants. In a few cases, linalool production in vegetative tissue in response to insect damage has been observed (Turlings et al., 1992), and it is
  • this compound may be used by the plants in such a response. It is contemplated, therefore, that the genes and proteins of the present invention may be useful in the protection of plants from insect damage. Purification and Characterization of LIS
  • chromatographical purification protocol was developed that employed anion exchange (DE52) column, HAP column, and another ion-exchange column, FPLC's Mono-Q.
  • a crude extract was prepared from 50 g of stigma tissue (derived from about 20,000 stigmata). The stigmata was used because they have the highest specific activity of LIS (subsequently, LIS was also partially purified from petals, and it appears to be an identical protein).
  • LIS protein was calculated to have a molecular weight of app. 83 kDa from its migration in the gel. On a
  • LIS activity elutes at about 70 kDa, indicating that LIS is active as a monomer.
  • N-terminal sequencing of LIS eluted from Mono-Q gave a clear sequence (with initial yield >50%) indicating no heterogeneity in the purified enzyme (22 residues were determined). In addition, the sequences of several internal fragments were determined.
  • the identity of the clones is determined in several ways. First, the oligonucleotide PCR probes are used as sequencing primers to determine the nucleotide sequence of adjacent regions in the clones. Because only parts of the amino acid sequences which had been obtained are used in the design of the oligonucleotides (7-8 residues out of 20-25 residues per peptide sequence), complete
  • concinna backcross progeny These plants are analyzed for emission of linalool and other volatiles.
  • the trait of linalool emission is somewhat quantitative, but two distinct classes of strong emitters and non-emitters are clearly evident. Although the quantitative nature of the trait complicates the genetic analysis, the working hypothesis is that for a progeny to be a strong linalool emitter it has to have inherited the C. breweri version of the Lis gene, and therefore the putative Lis cDNA clones are used as probes to carry out Southern blots of F 2 and backcross progeny DNA to look for co-segregation of the C. breweri Lis allele with the trait of strong linalool emission.
  • the preferred expression vector is pET, based on the T7 polymerase and promoter system (Studier et al., 1990).
  • concinna stigma LIS enzyme the differences in the levels of LIS activity among C. breweri stigma, petals, and C. concinna stigma are not believed to be due to changes in turn-over number or other kinetic parameters of the enzyme. On the other hand, these differences are strongly correlated with the differences in the amount of protein. Thus, it appears that C. breweri has evolved the ability to make (and emit) greater amounts of
  • Expression levels of the Lis gene in stigma and petals in the two species may be determined by first determining the steady-state level of the mRNA with the Northern blotting technique, using the Lis cDNA clone as a probe, and also by primer extension, a technique that also allows the determination of the start of transcription (Kellman et al ., 1990; Kellman et al., 1993; Piechulla et al., 1991). Run-on transcription assays may also be used to measures rates of
  • the nucleic acid segments of the present invention may also be used to isolate and characterize the Lis genes from various tissues in both species by screening genomic libraries constructed in the lambda vector EMBL3.
  • the promoter region of the C. breweri Lis gene may now be characterized by in vivo methods, such as reporter gene constructs, for example, and used for tissue specific expression of foreign genes . This may be done by the use of a
  • GUS promoterless reporter gene
  • GUS Jefferson, 1987
  • Several constructs may be made, each missing a certain part of the Lis promoter. These constructs would preferably be used to transform Nicotiana tabacum SRI plants, using the binary vector system of the Ti plasmid of Agrobacterium tumefaciena (An et al., 1986). Plants are regenerated, and optimal sequences for various promoter functions may thus be identified by the effect that their removal has on GUS activity (visualized by the addition of the GUS substrate to detached whole or sectioned organs).
  • hybridization technique may be used (Cox et al., 1988). The use of this technique is necessary because in plants, different types of cells may exist in a one-layer region and they cannot easily be separated in amounts large enough to extract mRNA.
  • the present invention allows one to perform immunofluorescence assays (Baskin et al., 1992) using anti-LIS antibodies disclosed herein.
  • the present invention comprises an antibody that is immunoreactive with a linalool synthase polypeptide.
  • An antibody can be a polyclonal or a monoclonal antibody and is preferably a monoclonal antibody.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal .
  • an immunogen comprising a polypeptide of the present invention
  • collecting antisera from that immunized animal .
  • a wide range of animal species can be used for the production of
  • an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a mouse with an LIS composition.
  • the spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the LIS polypeptide.
  • spleen cells are removed and fused, using a standard fusion protocol (see, e . g., The Cold Spring Harbor Manual for Hybridoma Development, incorporated herein by reference) with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against LIS .
  • Hybridomas which produce monoclonal
  • antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.
  • both poly- and monoclonal antibodies against LIS may be used in a variety of embodiments .
  • they may be employed in antibody cloning
  • protocols to obtain cDNAs or genes encoding LIS or related proteins may also be used in inhibition studies to analyze the effects of LIS in particular cells or tissue types.
  • a particularly useful application of such antibodies is in purifying native or recombinant
  • LIS for example, using an antibody affinity column, or screening for Lis expressing cells.
  • the operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, binding sites on
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • proiine (-1.6); histidine (-3.2); glutamate (-3.5);
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • threonine (-0.4); proiine (-0.5 ⁇ 1); alanine (-0.5);
  • valine (-1.5); leucine (-1.8); isoleucine (-1.8);
  • amino acids whose hydrophilicity values are within ⁇ 2 are preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • mutagenesis is well known in the art, as exemplified by various publications.
  • the technique may employ a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage .
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutagenic agents such as hydroxylamine
  • Volatile monoterpenoid production in individual flowers of four separate plants was monitored over a 7 d period beginning on the day before anthesis and
  • a crude protein extract was prepared by macerating flower parts in a microcentrifuge tube in the presence of ice-cold buffer (10 volumes : fresh weight) containing 50 mM BisTris-HCl pH 6.9, 10 mM DTT, 5 mM Na 2 S 2 O 5 , 1% (w/v) PVP-40 and 10% (v/v) glycerol.
  • the slurry was prepared by macerating flower parts in a microcentrifuge tube in the presence of ice-cold buffer (10 volumes : fresh weight) containing 50 mM BisTris-HCl pH 6.9, 10 mM DTT, 5 mM Na 2 S 2 O 5 , 1% (w/v) PVP-40 and 10% (v/v) glycerol.
  • the slurry was prepared by macerating flower parts in a microcentrifuge tube in the presence of ice-cold buffer (10 volumes : fresh weight) containing 50 mM BisTris-HCl pH 6.9, 10 mM
  • Linalool synthase activity was assayed by diluting 10 ⁇ L of crude extract (1.5-3 ⁇ g protein) into 80 ⁇ L of assay buffer (50 mM potassium HEPES pH 7.8, 5 mM DTT, 5 mM sodium metabisulfite, 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM MnCl 2 [Lewinsohn et al., 1991]), adding 10 ⁇ L of [1- 3 H]-GPP to final concentration 16 ⁇ M, at 150 mCi/mol [substrate synthesized according to Croteau and Cane (1985)], and overlaying the mixture with either pentane or hexane to trap volatile metabolites. The tube was then vortexed briefly and incubated at 20°C for 1 h.
  • assay buffer 50 mM potassium HEPES pH 7.8, 5 mM DTT, 5 mM sodium metabisulfite, 10% (v/v)
  • the first buds become visible on the C. breweri plant 4-6 weeks after germination.
  • the closed buds develop for approximately 12-18 d before the fused sepals split and reflex,
  • furanoid linalool oxide was emitted at much lower level, peaking at 0.4 ⁇ g/flower/12 h. Senescence (wilting) of all flowers was observed by the morning of Day 5, yet low levels of linalool and linalool oxides were emitted until abscission on the evening of Day 6.
  • Tissue levels of linalool peaked on the evening of Day 2 and then declined, paralleling the pattern found for linalool emission.
  • the maximal level of linalool found in the flower tissue was approximately 10% of the amount of linalool emitted by the flower over a 12 h period.
  • Tissue levels of linalool oxides peaked at Day 3, while the emission of linalool oxides was highest at Day 2. On Day 2, levels of linalool oxides in the tissue
  • C. concinna flowers C. concinna, a close relative of C. breweri, has flowers which are smaller and which do not emit a
  • LIS activity was found in several parts of the flowers. At the peak of LIS activity, at Days 1-2, total LIS activity was similar in petals and pistil (stigma + style) . However, stigmata possessed the highest level of LIS activity per fresh weight, followed by style tissue (with 25-35% of the specific activity found in stigma), petals (20% of stigma LIS specific activity at peak time) and stamens. None of the remaining floral parts -sepals, hypanthium and ovaries - were found to contain any LIS activity. The vegetative parts of the plant were also devoid of LIS activity. Levels of LIS activity varied during the lifespan of the flower in concert with the levels of linalool
  • LIS activity was observed during Days 1-2. When flowers were pollinated on Day 3, LIS activity decreased by 90% in the stigma and 50% in the petals within 48 h, similar to the reduction in monoterpene emission following pollination. However, when pollination did not occur,
  • the level of LIS activity was investigated in buds. In order to carry out these studies, it was necessary to devise a method to classify stages in bud development. Due to variation in bud size and maturation time within and among plants, it was difficult to predict the exact number of days remaining before anthesis by bud size alone. However, in contrast to bud size, color changes during the development of the petals and stigma were found- a progression from white to dark purple - to be reliable markers for the
  • LIS activity in extracts of C. concinna flower parts of Day 2 flowers was examined. Consistent with the emission data (Tables I, II), LIS activity was detected only in the stigma, which had a level of activity of 2.8 fkat/stigma (and per flower) and 4.6 fkat/g fresh weight. These levels are respectively 0.3% of the LIS activity per stigma (0.01% per flower) and 3% per fresh weight of activity in C. breweri stigmata of the equivalent stage.
  • monoterpenes are present, they might not contribute to scent emission, or their contribution might not be essential.
  • the pistil is the only part of the flower (excluding the ovaries) that continues to increase in size and weight after the flower opens, but its specific LIS activity does not decrease, indicating that additional LIS activity accrues there, at least during the first few days after anthesis.
  • the petals constitute the bulk of the LIS-containing floral tissue (Table III) , and they possess a similar or even higher total amount of LIS activity compared with the pistil.
  • Each flower part that emits linalool or linalool oxides - petals, pistils and stamens - also contains significant LIS activity, whereas flower parts that do not contain LIS activity do not contain or emit these monoterpenes.
  • buds of later stages contain appreciable amounts of LIS activity but they do not accumulate or emit these monoterpenes. It is likely that earlier steps in the pathway are not yet operating in the buds, either for lack of other enzymes or because of sequestration of enzymes and/or substrates in
  • monoterpenes may be synthesized in buds of later stages but may be rapidly converted to other compounds or derivatives.
  • Crude protein extracts were prepared by homogenizing freshly excised stigmata in a chilled mortar in the presence of ice-cold buffer (10:1 (v/w) buffer: tissue) containing 50 mM potassium BisTris, pH 6.9, 10 mM dithiothreitol, 5 mM Na 2 S 2 O 5 , 1% (w/v) polyvinylpyrrolidone (Sigma, PVP-40) and 10% (v/v) glycerol. The slurry was passed through miracloth and centrifuged for 10 min at 12,000 g to produce a supernatant that contained the bulk of the LIS activity.
  • ice-cold buffer (10:1 (v/w) buffer: tissue) containing 50 mM potassium BisTris, pH 6.9, 10 mM dithiothreitol, 5 mM Na 2 S 2 O 5 , 1% (w/v) polyvinylpyrrolidone (Sigma, PVP-40) and 10% (v/v)
  • the crude extract (100 ml) was loaded onto a DEAE-cellulose (0.7 cm diam. x 6 cm) column (Whatman, DE52) that was pre-equilibrated with a solution containing 50 mM BisTris, pH 6.9, 10% glycerol and 10 mM DTT (buffer A) at a flow rate of about 1 ml/min.
  • the column was washed with 50 ml of buffer A followed by an additional 20 ml of buffer A containing 150 mM C1 .
  • LIS activity was then eluted with 20 ml of buffer A containing 250 mM KCl .
  • the column was washed with 20 ml buffer A and then eluted using a linear gradient (100 ml) from 0 to 200 mM Na-phosphate in buffer A.
  • the fractions containing LIS activity, eluting at about 100 mM Na-phosphate, were pooled (10 to 16 ml) and loaded on a pre-packed Pharmacia Mono-Q HR 5/5 FPLC column equilibrated with buffer A.
  • a 20-ml wash with buffer A a steep linear gradient from 0 to 100 mM KCl in buffer A (10 ml) was applied, followed by a more gradual linear gradient from 100 to 400 mM KCl in buffer A (100 ml, 0.25 ml/min).
  • the enzyme consistently eluted at about 220 mM KCl. After dialysis to assay conditions, the enzyme at this level of purity was used for
  • a Superdex 75 Hi Load 16/60 column (Pharmacia FPLC) was employed to determine the native molecular mass of the C. breweri linalool synthase. Hydroxyapatite-purified linalool synthase (1 ml, 44.1 pkat/mg protein) was loaded on the Superdex 75 column and eluted with buffer A containing 5 mM DTT, using a flow rate of 1 ml/min. Two-ml fractions were collected. Protein standards of known molecular mass (Sigma, St. Louis, MO) were used for calibration. For SDS-PAGE, the method of Laemmli (Laemmli, 1970) was employed, with 10% acrylamide and a ratio of 30:0.8 w/w acrylamide:bis-acrylamide.
  • LIS activity was determined by mixing 10 ⁇ l of the enzyme sample with 12 ⁇ M [1- 3 H]-GPP (s.a. 150 Ci/mol) in 100 ⁇ l of assay buffer (50 mM Hepes-KOH, pH 7.8, 10 mM DTT, 5 mM Na 2 S 2 O 5 , 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM MnCl 2 (Lewinsohn et al., 1991) and, as an overlay to trap volatiles, either 1 ml hexane (for scintillation counting) or 2 ml of pentane (for radio-GC analysis). The mixture was then vortexed briefly, centrifuged to separate phases (5 sec. Eppendorf) and incubated at 20°C without shaking for up to 1 h (higher temperatures resulted in elevated rates of chemical conversion of GPP to various alcohols, including linalool) . After
  • the initial column temperature was 170°C and, following a 5 min hold, was programmed at 5°C/min to 220°C, using He at a flow rate of 50 ml/min.
  • Control incubations included the omission of enzyme extract, the use of boiled enzyme extract, the omission of substrate, and the omission of divalent cations. All of these controls produced negligible radioactivity in the hexane phase of the enzyme assay mixtures.
  • Protein was determined by the method of Bradford (Bradford, 1976), using bovine serum albumin as standard.
  • the enantiomeric composition of linalool samples was determined on a fused silica capillary column (0.25 mm internal diam. ⁇ 30 m) coated with a 0.25 ⁇ m film of ⁇ -cyclodextrin (J & W Scientific, Cyclodex B) , operated at an initial temperature of 75° C for 15 min followed by a rise of 5°C/min to 200° C. H 2 at a head pressure of 13.5 psi was used as carrier gas (Alonso et al., 1992).
  • the native molecular mass of linalool synthase activity was determined to be about 73 ⁇ 5 KDa based on co-elution with bovine serum albumin on gel permeation chromatography and comparison of the elution volume to those of known proteins (FIG. 3). Based on a subunit mass of 79 KDa determined by SDS-PAGE (FIG. 2) and a native molecular size of 73 KDa indicated by gel
  • linalool synthase is thought to be a monomer of 76 ⁇ 3 KDa.
  • Other monoterpene synthases from higher plants are active monomers in the 50-70 KDa range (Alonso, 1992; Lewinsohn et al ., 1992b), or homodimers with 40-50 KDa subunits (Gambliel et al., 1984).
  • the preparations were devoid of other monoterpene synthase activities or GPP phosphohydrolase activity as evidenced by the absence of [ 3 H]-geraniol in the reaction products.
  • C. breweri leaves contained only GPP phosphohydrolase activity, as leaf-derived cell-free extracts produced only geraniol.
  • the enantiomer compositions of the linalool emitted by C. breweri flowers obtained by head space collection
  • S-linalool synthase requires a divalent metal ion for activity.
  • a K m value of 0.9 ⁇ M was calculated for GPP from a Lineweaver-Burk plot. This value is in the range
  • the enzyme is unstable in the absence of DTT (>95% loss over 2 h at 4 °C), and its later addition does not result in the regaining of activity.
  • Triton-X-100 0.1% w/v
  • the enzyme is very stable to vigorous vortexing in the presence of non-polar organic solvents such as hexane and pentane.

Abstract

L'invention porte sur un polypeptide purifié de synthase de S-linalol à partir de Clarkia breweri, représentant le polypeptide recombiné et les séquences d'acide nucléique codant ledit polypeptide. L'invention concerne également des anticorps immunoréactifs au peptide purifié et aux versions recombinées dudit polypeptide. L'invention concerne enfin des procédés utilisant lesdites séquences d'acide nucléique, et des procédés de stimulation de l'odeur et du goût de plantes exprimant lesdites séquences d'acide nucléique.
PCT/US1996/016482 1995-10-12 1996-10-15 Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs WO1997015584A2 (fr)

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AU76632/96A AU7663296A (en) 1995-10-12 1996-10-15 Use of linalool synthase in genetic engineering of scent production

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JP2013013406A (ja) * 2011-06-10 2013-01-24 Suntory Holdings Ltd リナロール合成酵素をコードするポリヌクレオチドおよびその用途
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WO1998050570A2 (fr) * 1997-05-08 1998-11-12 The Regents Of The University Of Michigan Procedes et compositions ayant trait a l'utilisation d'(iso)eugenol methyltransferase
WO1999023226A1 (fr) * 1997-10-30 1999-05-14 The Regents Of The University Of Michigan Procedes et compositions pour l'utilisation de benzylalcool acetyl transferase
US6559297B2 (en) 1998-09-18 2003-05-06 The Salk Institute For Biological Studies Synthases
US6890752B2 (en) 1998-09-18 2005-05-10 The University Of Kentucky Research Foundation Synthases
US6569656B2 (en) 1998-09-18 2003-05-27 The University Of Kentucky Research Foundation Synthases
US6468772B1 (en) 1998-09-18 2002-10-22 The Salk Institute For Biological Studies Methods of making modified polypeptides
US6495354B2 (en) 1998-09-18 2002-12-17 University Of Kentucky Research Foundation Synthases
WO2000050613A2 (fr) * 1999-02-22 2000-08-31 Yissum Research And Development Company Of The Hebrew University Of Jerusalem Plantes transgeniques et procede de transformation d'oeillets
WO2000050613A3 (fr) * 1999-02-22 2000-12-14 Yissum Res Dev Co Plantes transgeniques et procede de transformation d'oeillets
US7129393B1 (en) 1999-02-22 2006-10-31 Yissum Research Development Company Of The Hebrew University Transgenic plants and method for transforming carnations
EP1130104A1 (fr) * 2000-02-16 2001-09-05 Stichting Dienst Landbouwkundig Onderzoek Réduction de la dégradation des produits végétales in planta
WO2001061023A1 (fr) * 2000-02-16 2001-08-23 Plant Research International B.V. Reduction de la degradation in planta de produits d'une plante recombinante
WO2002064764A2 (fr) 2001-02-12 2002-08-22 Plant Research International B.V. Synthases d'isoprenoides
WO2002064764A3 (fr) * 2001-02-12 2003-12-31 Plant Res Int Bv Synthases d'isoprenoides
EP1231273A1 (fr) * 2001-02-12 2002-08-14 Plant Research International B.V. Terpene synthase/cyclase et olefin synthase et leur utilisation
US7453024B2 (en) 2001-02-12 2008-11-18 De Ruiter Seeds R&D B.V. Isoprenoid synthases
US8338663B2 (en) 2001-02-12 2012-12-25 Monsanto Invest N.V. Isoprenoid synthases
EP1377156A1 (fr) * 2001-03-22 2004-01-07 Scentgene Pollination Ltd. Procede pour accroitre l'entomophilie
EP1377156A4 (fr) * 2001-03-22 2007-08-29 Scentgene Pollination Ltd Procede pour accroitre l'entomophilie
WO2008077986A1 (fr) * 2006-12-27 2008-07-03 Consejo Superior De Investigaciones Científicas Amélioration de la teneur aromatique des vins et autres boissons alcoolisés moyennant l'utilisation de micro-organismes produisant, en cours de fermentation, des monoterpène synthases
ES2324508A1 (es) * 2006-12-27 2009-08-07 Consejo Sup. De Invest. Cientificas Mejora del contenido aromatico de vinos y otras bebidas alcoholicas mediante la utilizacion de microorganismos que, durante la fermentacion, producen monoterpeno sintasas.
JP2013013406A (ja) * 2011-06-10 2013-01-24 Suntory Holdings Ltd リナロール合成酵素をコードするポリヌクレオチドおよびその用途
CN110452918A (zh) * 2019-08-30 2019-11-15 福建农林大学 一种创制高香型矮牵牛新种质的分子方法

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