WO2000055303A2 - Plantes transgeniques possedant des proprietes de saveur accruees - Google Patents

Plantes transgeniques possedant des proprietes de saveur accruees Download PDF

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WO2000055303A2
WO2000055303A2 PCT/US2000/007330 US0007330W WO0055303A2 WO 2000055303 A2 WO2000055303 A2 WO 2000055303A2 US 0007330 W US0007330 W US 0007330W WO 0055303 A2 WO0055303 A2 WO 0055303A2
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plant
transgenic
nucleic acid
levels
cgs
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WO2000055303A3 (fr
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Nilgun E. Tumer
Thomas Leustek
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Rutgers, The State University
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Priority to US09/936,454 priority patent/US6821781B1/en
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Publication of WO2000055303A3 publication Critical patent/WO2000055303A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/8242—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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates generally to the field of agricultural biotechnology and more particularly to transgenic plants exhibiting enhanced flavor quality and stability, and methods of making the plants.
  • Methionine (Met) is the precursor for methional, an important flavor compound in various plants such as potatoes, as well as in meats and cheese cracker flavors.
  • the production of methional is thermally induced. It is formed as a result of the interaction of alpha-dicarbonyl compounds, which are formed during the Maillard reaction, with Met through the Strecker degradation reaction. Methional readily decomposes to yield methanethiol, which oxidizes to dimethyl disulfide.
  • derivatives of methionine such as S- methylmethionine, release dimethyl sulfide that is responsible for the aromas of fish, canned sweet corn, tomato juice and stewing oysters and clams.
  • Methional and S-methyl methionine are heat labile and readily decompose during food processing. Due to high costs of production, these flavor compounds as well as the Met precursor are not added back during food processing.
  • Met a sulfur containing amino acid is extremely important in all living organisms. Met serves central roles in metabolism as the initiator tRNA in protein synthesis, as S-adenosylmethionine (SAM), the primary methyl donor for most transmethylation reactions and as a precursor for polyamines and the phytohormone, ethylene in plants. Animals cannot synthesize Met, for this reason it is considered an essential amino acid for animal nutrition.
  • SAM S-adenosylmethionine
  • BNP Brazil Nut
  • Lys level was significantly reduced in mature seeds because Lys is efficiently catabolized (Karchi, et al, supra). Further analysis revealed that a Lys degradation pathway was induced in the transgenic plants (Galili, et al, Plant Cell 7:899-906 (1995)).
  • DHPS dihydrodipicolinate synthase
  • Solanaceous family e.g., tomato, potato and eggplant, containing at least one non-native nucleic acid that when expressed in the plants, results in increased free methionine levels relative to native free methionine levels.
  • Processing of edible portions of the plant e.g., to make a food product or food additive containing the processed plant portion(s), results in an increase in methional levels compared to methional levels present in a processed edible portion of a wild-type plant.
  • the non-native nucleic acid encodes cystathionine gamma synthase (CGS).
  • increased free methionine levels in potatoes result from expression of a nucleic acid containing a tuber-specific promoter regulatory unit operably linked (in operative association with) and anti-sense S-adenosyl-methionine synthetase (SAMS)-encoding nucleic acid.
  • SAMS S-adenosyl-methionine synthetase
  • a non-native nucleic acid containing a tuber-specific promoter linked to a DNA molecule encoding a SAMS DNA in the sense orientation that is homologous to the potato. Seed derived from the transgenic plants are also provided.
  • Another aspect of the present invention is directed to transgenic plants having edible portion(s) that produce methional when processed, such as maize and soybean plant, that contain increased free methionine levels relative to native free methionine levels.
  • Maize is especially preferred.
  • Edible portions of these plants contain increased methional levels compared to methional levels in a processed, wild-type plant.
  • the increased free methionine levels are achieved by expression of a non-native nucleic acid that is other than a nucleic acid encoding a plant CGS, particularly in the seeds of the plants.
  • Yet another aspect of the present invention is directed to methods of making the aforesaid transgenic plants.
  • a further aspect of the present invention is directed to making processed products such as foods or food additives containing edible parts thereof that exhibit increased flavor stability and/or quality.
  • the methods entail preparing the aforesaid transgenic plants that contain increased methionine levels, and harvesting and then processing the plant parts, whereupon the processing results in increased methional levels. Products such as foods and food additives that contain the processed plant or parts thereof are also provided.
  • Yet a further aspect of the present invention is directed to a method for selecting plant cells containing a non-native nucleic acid of interest (e.g., a structural gene encoding a protein of interest), and a selection agent/marker gene combination for use therewith.
  • a non-native nucleic acid of interest e.g., a structural gene encoding a protein of interest
  • the method entails transforming plant cells with a chimeric nucleic acid that contains inoperable association, a promoter functional in a plant cell and a first and a second DNA molecule.
  • the first DNA molecule is preferably a structural gene encoding a protein of interest and the second DNA molecule encodes a CGS that permits the selection of a transformed plant cell containing the chimeric nucleic acid molecule by rendering the transformed plant cell resistant to an amount of ethionine that would be toxic to a plant cell that does not express the DNA encoding the CGS.
  • the transformed plant cells are cultured in medium containing ethionine in an amount that would be toxic to plant cells that do not express the CGS-encoding DNA. Plant cells that grow in the medium are selected.
  • the chimeric nucleic acids, plant cells transformed therewith, and compositions of matter containing the transformed plant cells in medium containing ethionine are also provided.
  • Fig. 1 is a flow diagram showing the biosynthetic pathway of methionine in plants, wherein ACC means 1-aminocyclopropane-l-carboxylic acid, CGS means cystathionine gamma-synthase, DMS means dimethylsulfide, SMSP means dimethylsulfoniopropionate, Hey means homocysteine, Kan® means kanamycin resistant, MTA means 5 -methyl thioadenosine, MTHB means 4-methylthio-2-hydroxy butryic acid, MTOB means 4-methylthio-2-oxobutanoic acid, OPH means O-phosphohomoserine, PITC means phenylisothiocyanate, SAH means S-adenosyl-L-homoserine, SAM means S-adenosyl- L-methionine, SAT means serine acetyltransferase, SMM means S-methylmethionine
  • Fig. 3 is a bar graph showing the content (in pmol/mg) of the free amino acids Met, Thr and Tyr in tubers of wild-type and transgenic plants expressing Arabidopsis CGS;
  • Fig. 4 is a bar graph showing the content (in pmol/mg) of the free amino acids Met, Thr and Tyr in roots of wild-type and transgenic plants expressing Arabidopsis CGS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Met along with threonine (Thr), lysine (Lys) and isoleucine (He) are derived from aspartic acid (Asp).
  • the pathway for Met synthesis varies among different organisms.
  • the first step in the pathway is the phosphorylation of Asp by the enzyme aspartokinase to generate O-phospho-L-homoserine (OPH).
  • O-phospho-L-homoserine O-phospho-L-homoserine
  • CGS cystathionine- ⁇ -synthase
  • cysteine cysteine
  • the second enzyme in the pathway cystathionine- ⁇ -lyase, cleaves cystathionine to form L-homocysteine and L-serine. Met is then formed by transmethylation of L-homocysteine.
  • the enzymes involved in the Met biosynthetic pathway are distributed between plastids and cytosol. CGS is localized exclusively in the chloroplasts.
  • Free Met levels are increased (relative to wild-type or non-transformed plant) by over-expressing or inhibiting expression of enzymes involved in its biosynthetic pathway.
  • the present invention is particularly suited to members of the Solanaceous family, e.g., potato, tomato and eggplant, as well as to other plants that produce methional when processed, such as maize and soybean .
  • over-expression of CGS or the inhibition of S-adenosyl- methionine synthetase (SAMS) or threonine synthase will increase free Met levels.
  • a transgenic plant is prepared that over-expresses CGS.
  • DNAs encoding this enzyme have been isolated and sequenced from a variety of plant species including ice plant, potato, maize (WO/9531554), soybean, spinach (Ravanel, et al, Arch. Biochem. Biophys. 375:572-584 (1995)) and tobacco (Steegbora, et al, J. Mol. Biol. 290 5J:983-996 (1999) and Arabidopsis (Kim, et al, Plant Mol. Biology 32:1117-1124 (1996)), as well as yeast, bacteria and algae.
  • CGS genes from other plant species can be identified by constructing a plant cDNA library in an E.
  • the potato CGS genes show 93% sequence similarity as well as high sequence similarity with other plant CGS genes, including Arabidopsis.
  • the N-terminal region of the potato CGS genes contain typical features of transit peptides for localization to plastids and the conserved region of MTO1 which has been shown to regulate CGS expression in Arabidopsi .
  • Chiba describes five different CGS alleles in Arabidopsis (i.e., Mtol-1, Mtol-2, Mtol-3, Mtol-4 and Mtol-5) resulting from point mutations that were found to be concentrated in a region of .exonl of the CGS gene termed MTO1. This region consists of approximately 30 amino acids that are highly conserved among plant CGS sequences.
  • MTO1 a region of .exonl of the CGS gene termed MTO1.
  • This region consists of approximately 30 amino acids that are highly conserved among plant CGS sequences.
  • Application of exogenous free Met to wild-type plants results in a decrease in CGS mRNA. The deduction, therefore, is that CGS normally regulates its own synthesis in plants.
  • mRNA synthesis decreases with increasing levels of free Met.
  • the mtol mutants described in Chiba over-accumulate free Met, indicating CGS mRNA levels were not down-regulated in the presence of free Met.
  • the mutations give rise to amino acid substitutions near the N-terminus of each of the 5 encoded CGS proteins, namely Gly84Ser, Ser ⁇ lAsn, Gly84Asp, Arg77His and Gly84Asp respectively. These mutations can be introduced into the wild-type Arabidopsis CGS gene using site-directed mutagenesis, thus producing DNAs encoding mutant, non-self regulating CGS.
  • SAMS catalyzes the synthesis of S-adenosyl methionine (SAM), a common methyl donor in various methylation reactions, from Met and ATP. This reaction consumes two high energy phosphate bonds of ATP releasing pyrophosphate.
  • SAM S-adenosyl methionine
  • Met is a substrate of SAMS and SAMS activity results in a decrease in free Met levels.
  • SAMS from E. coli is a homotetramer consisted on four 383-residue monomer (Markham, et al, J. Biol. Chem.
  • Plant SAMS sequences have been reported from various plant species. In all cases examined, SAMS was found to be encoded by small gene family. There are 2 genes found in Arabidopsis (Peleman, et al, Plant Cell 7:81-93 (1989), 4 genes in tomato ( ⁇ spartero, et al, Plant Mol. Biol. 25(2):2ll-221 (1994)), and 3 genes in Catharanthus roseus (Schroder et al, Plant Mol. Biol. 33:211-222 (1997)).
  • SAMS is encoded by a gene family present in most angiosperms.
  • One gene is expressed in vascular tissue of roots, stems and leaf, and the other is expressed in mesophyl cells of roots stems and leaf.
  • the SAMS expressed in vascular tissue may function in supplying S- adenosylmethionine, the primary methyl group donor, for lignin biosynthesis in vascular cells with extensive secondary cell wall synthesis. Both forms encode cytosolic enzymes.
  • the form expressed in mesophyll cells may provide S-adenolsylmethionine for other metabolic reactions including polyamine biosynthesis and others.
  • SAMS expression is partially repressed using antisense DNA.
  • This technology involves expression of a transgene, cloned in reverse (or anti-sense) orientation relative to a promoter sequence for regulating transcription of the DNA.
  • the anti- sense transcript is believed to hybridize to the endogenous sense transcript, blocking its translation and targeting it for degradation.
  • the important features of this technology are that only transcripts with significant nucleotide sequence similarity to the anti-sense are targeted for repression, and the repression is usually incomplete.
  • the effect of the anti-sense ranges from nearly complete repression to slight repression. In so doing, specific SAMS isoenzymes may be repressed, specifically in potato tubers.
  • an SAMS anti-sense nucleic acid is operably linked to a tuber specific promoter.
  • a preferred promoter is the patatin promoter.
  • Other such promoters in their native state, regulate transcription of mRNA encoding proteins involved in starch biosynthesis that occurs in tubers.
  • partial suppression of SAMS in tubers is achieved via co-suppression wherein a SAMS-encoding nucleic acid is operably linked to a potato tuber specific promoter.
  • the trans-nucleic acid is preferably homologous to the plant and embraces tuber-specific over-expression of the native SAMS gene.
  • non- native it is meant different from the native genome or in addition to the native genome of the plant.
  • Bacterial CGS genes may also be used in the present invention.
  • Bacterial CGS has been well studied and is known to use 0-succinylhomoserine (OSH) or 0- acetylhomoserine (OAH), depending on the species, as the physiological substrate.
  • OSH 0-succinylhomoserine
  • OAH 0- acetylhomoserine
  • the bacterial enzyme catalyzes a ⁇ -elimination reaction in the absence of Cys to yield succinate, ⁇ - ketobutyrate and ammonia.
  • the nucleic acid sequence and these various properties of bacterial CGS are reported in Holbrook et al, Biochem. 75:435-442 (1990).
  • Another method involves co-expression of a methionine-rich protein and a protease in different cellular compartments. Upon potato processing, the protease is allowed to come into contact with the Met-rich protein, cleaving it into free Met residues. Yet another method entails expressing an anti-sense DNA encoding threonine synthase (TS) or DNAs encoding mutant TSs that inhibit or reduce the amount of TS synthesized by the plant cell. Free Met levels increase when TS is inhibited or reduced.
  • TS threonine synthase
  • the present invention is also applicable to plants other than members of the Solanaceous family that produce methional when processed. These plants include maize and soybean plants. Applicants have discovered that processed edible parts of such transgenic plants having relatively high free Met levels conpared to wild type plants have significantly improved flavor stability and/or quality and nutritional quality relative to processed parts obtained from plants containing native methional levels. Transgenic plants are prepared preferably using techniques described above other than a plant CGS-encoding transgene and less preferably, any CGS gene. In the case of soybeans and maize, the non-native nucleic acid e.g., the anti-sense SAMS, is preferably expressed in the seeds, which is the edible part of these plants that is processed.
  • the non-native nucleic acid e.g., the anti-sense SAMS
  • a preferred method for dicots entails indirect gene transfer with Agrobacterium tumefaciens-modiated transformation of potato tissue, and regeneration of the transformed tissue into whole plants.
  • Preferred methods of direct gene transfer into monocots include PEG- mediated gene transfer and particle bombardment. Seed may be derived from the plants in accordance with standard methods.
  • Processing of the plant such as potato results in increased levels of methional relative to plants having native or wild type levels of free Met, which in turn results in increased flavor and nutritional quality.
  • processing is meant to include cooking, dehydrating, baking and other activities that are performed to prepare various plant products and/or parts thereof for human consumption.
  • the edible portion is the tuber and such products include frozen (freeze dried) potato products, boiled or baked potato, mashed potato, escalloped potato, potato chips and types of fried potato (e.g., french fries).
  • the edible portion is the fruit and such products include processed tomatoes, canned tomato products such as pastes, sauces, juices, and the like.
  • Processed corn products include corn fries, canned corn etc.
  • Literature directed to methods for making food products using plants and plant parts is legion. See, e.g., U.S. Patents 5,952,026 and 5,965,591 (processed potato products), U.S. Patents 5,902,616, 6,004,591 and 5,965,190 (processed tomato products) and U.S. Patents 5,968,585 and 5,928,701 (processed corn products).
  • Another aspect of the present invention is directed to a marker gene/selection agent combination that is used to identify cells transformed with a nucleic acid of interest.
  • Ethionine is an analog of Met that is very toxic to plants. Applicants have discovered that plants that over-produce CGS are resistant to CGS. Accordingly, this aspect of the present invention entails use of the CGS/ethionine selection system for plant cells much in the same way that popular selection systems e.g., neomycin/NPTII and BASTA Bargene, are used.
  • the CGS DNA is incorporated into an expression cassette that also includes the nucleic acid of interest.
  • a nucleic acid encodes a protein e.g., a protein that confers a phenotypic change to the plant.
  • cells are cultured on medium containing ethionine.
  • the amount of methionine added to the medium varies depending upon the type of plant cells being cultured, and may be determined by persons skilled in the art in accordance with standard techniques. In Example 1 below, concentrations of ethionine ranging from 10-300 ⁇ M were added to cultures of transformed potato cells. Cells/protoplasts that grow in the presence of ethionine are selected.
  • CGS is a plant enzyme
  • NPTII bacterial neomycin phosphotransferase
  • the present method is applicable to any plant cell, including both monocots and dicots.
  • monocots are transformed using particle bombardment or via PEG-mediated uptake, and dicots are transformed using Agrobacterium tumafaciens-mQdiatQd gene transfer.
  • the products and methods of the present invention are further illustrated by the following examples. The presentation of these examples is by no way intended to limit applicants' invention in any way. Unless otherwise specified, all percentages are by weight.
  • Example 1 Constitutive Expression of Arabidopsis CGS in Potato
  • the following example describes the isolation of a cDNA clone encoding CGS from Arabidopsis thaliana complementation of a CGS mutant strain of E. coli (met B) (Kim, et al, (1996).
  • CGS is a single copy gene in Arabidopsis.
  • the coding sequence is 1692 bp and encodes a 563 amino acid protein.
  • the cDNA encoding the Arabidopsis CGS was expressed in Russet Burbank potato.
  • the transgenic potato plants were analyzed for expression of CGS, CGS activity, and free methionine levels. Preparation of the CGS[+] construct
  • a plant expression vector was prepared for over-expression of CGS in transgenic potato plants by subcloning the 2.0 kb Kpnl-Xbal fragment from the full-length CGS cDNA (GenBank Accession number U43709; Kim, et al, Plant Mol. Bio. 32(6):X X X1- 1124 (1996)) into the same sites of pFF19 as a transcriptional fusion with the CaMV 35S promoter.
  • the entire expression cassette was subcloned as a 3.0 kb H/ « ⁇ 7111-EcoRI fragment into same sites of pBHOl (Clonetech) in the sense orientation CGS[+]. Transformation of potato The CGS[+] construct was used to transform A.
  • tumefaciens strain pGV2260 by electroporation BioRad, Inc., Gene PulserTM.
  • Ten milliliters of overnight-grown A. tumefaciens was inoculated into 1 L of YEP medium (for 1 L, 10 g of Bacto-peptone, lOg of Bacto-yeast extract, and 5 g of NaCl, p ⁇ 7.0) and the culture was grown to an OD600 of 0.4 at 30 °C.
  • the cells were harvested at 4 °C by centrifugation at 4000 g for 10 min. and resuspended in 1 L of ice cold water. The process was repeated once more.
  • tumefaciens strain pGV2260 carrying the CGS[+] construct using cultured potato stems as explants. 8-10 mm long stems were co-cultivated with Agrobacterium carrying CGS [+] resuspended in water. The stems were then placed on petri dishes containing MSO medium (MS, 3% sucrose and lx vitamin) for two days in the dark. They were then transferred to PC medium (MS salts with 100 mg/1 myo-inositol, 0J mg/1 NAA, 3% sucrose, 5 mg/1 AgNO3, 0.5 mg/1 zeatin, 100 mg/1 kanamycin for selection and 300 mg/1 cefataxime to prevent contamination) for the induction of callus tissue.
  • MSO medium MS, 3% sucrose and lx vitamin
  • PC medium MS salts with 100 mg/1 myo-inositol, 0J mg/1 NAA, 3% sucrose, 5 mg/1 AgNO3, 0.5 mg/1 zeatin, 100 mg/1 kanamycin for selection
  • the tissues were placed on PS medium (MS, 100 mg/1 inositol, lx vitamin, 3% sucrose, 0.3 mg/1 gibberellic acid, 5 mg/1 zeatin, 100 mg/1 kanamycin and 300 mg/1 cefataxime) to induce shoots.
  • PS medium MS, 100 mg/1 inositol, lx vitamin, 3% sucrose, 0.3 mg/1 gibberellic acid, 5 mg/1 zeatin, 100 mg/1 kanamycin and 300 mg/1 cefataxime
  • PM medium MS, 3% sucrose, 170 mg/1 NaH 2 PO 4 -H 2 O, 0.4 mg/1 thiamine, 100 mg/1 myo-inositol and lx vitamin
  • the plantlets were then transferred to soil and acclimatized to the greenhouse conditions.
  • Genomic DNA was isolated from potato plants using a modified purification method. Plant tissues were ground in liquid nitrogen and resuspended in urea extraction buffer (2.8 M urea, 0.125 M NaCl, 20 mM Tris-HCl, pH 8.0, 8 mM EDTA, 0.4% sarkosyl (a detergent). After extraction with phenol/chloroform, genomic DNA was precipitated by iso- propyl alcohol and resuspended in TE buffer. Total RNA was isolated using TRI REAGENT (T-9424, Sigma) according to the manufacturer.
  • CGS protein levels were determined in transgenic potato plants by immunoblotting as previously described (Wang, et al, Plant Physiol. 702:843-850 (1993)). Soluble protein extracts were prepared from leaves, roots and tubers of transgenic potato plants by grinding the plant tissues in liquid nitrogen and resuspending the powder in 50 mM Tris- HCI, pH 8.0. The homogenate was centrifuged at 4 °C to remove cellular debris, and the final supernatant was used for immunoblot analysis. Protein concentrations were determined using the Bradford dye-binding assay with BSA as a standard (BioRad, Inc.).
  • Protein samples (10 ⁇ g) were electrophoresed on an SDS-PAGE gel prepared with 10% (w/v) acrylamide and blotted onto Immobilon-P membrane (Millipore Corp.) at 4 °C for 1 hr. using transfer buffer containing 25 mM Tris, 250 mM glycine and 20% (v/v) methanol. The membrane was blocked with 1% (w/v) blocking reagent (Boehringer Mannheim) overnight at 4 °C. Antiserum against Arabidopsis CGS was used at a dilution of 1 :2000 in PBST (0.1 M phosphate buffer pH 1.4, 15 mM NaCl, 3 mM KC1, 0.2% (v/v) Tween-20).
  • the membrane was incubated with antibodies for 1 hr. and then the membrane was washed with PBST 3 times for a total washing time of 1 hr. The membrane was then incubated with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma, A0545) diluted 1 :10000 in PBST buffer for 30 min. After washing with PBST 3 times for total of 30 min., the antibody complexes were detected with the Renaissance Kit (Dupont NEN, Inc.) and Kodak X-OMATTM film (Eastman Kodak Com., NY). CGS enzyme assay
  • Enzyme activity was measured in a volume of 100 ml containing 20 mM Mops-NaOH, pH 1.4, 0.5 mM Cys, 10 mM OSH, 1 mM DTT and plant extracts. Assays were initiated by adding substrate. After incubation at 20 °C for 2 to 30 min., the reaction was stopped by addition of 50 ml 20% (w/v) TCA. Cystathionine in the supernatant was measured by HPLC after derivatization with O-phthaldialdehyde (OP A) in the presence of b-mercaptoethanol. Twenty ml of the alkylation reaction was injected onto an RP-8 (10 mm particle size, LiChrosorb) column.
  • RP-8 10 mm particle size, LiChrosorb
  • Buffers used for elution of the OPA fluorescent derivatives were as follows: A, 85 mM sodium acetate and 6% (v/v) acetonitrile (pH 4.5); B, 60% (v/v) acetonitrile. The following linear gradients were used: 40 to 80% B, 0 to 6 min.: 80% B, 6 to 9 min.; 80 to 40% B, 9 to 10 min.; 40% B, 10 to 12 min. (40C, 1 ml min.).
  • the OPA derivatives were detected by measuring fluorescence at 455 nm after excitation at 340 nm using a Hitachi F-104 ⁇ fluorescence detector. Quantitation of cystathionine was carried out by measuring peak areas using the chromatography data analysis software Beckman SYSTEM GOLD. One unit of activity is defined as the formation of 1 mmol of cystathionine per minute. Amino acid analysis
  • Soluble amino acids from transgenic potato plants were measured by HPLC analysis of tissues extracted with ethanol (Inaba, et al, Plant Physiol. 704:881-887 (1994)) alkylated with phenylisothiocyanate (PITC) (Fierabracci, et al, J. Chromatog. 570f2 :285-291 (1991)). Plant samples were ground in liquid nitrogen. Powdered tissue was extracted with 80% (v/v) ethanol. The sample was centrifuged to remove cellular debris. The pellet was re- extracted twice more and the supernatants were pooled. The pooled extracts were loaded on an AG-50W (H+ form, BioRad) column (1.5 ml/mg fresh weight).
  • Russet Burbank potato plants were transformed with Agrobacterium tumefaciens carrying the Arabidopsis CGS gene under the control of the 35S promoter (CGS+ vector). Ten different transgenic lines were generated. All transgenic lines were phenotypically normal and indistinguishable from the untransformed potato plants. The genomic DNA from the 10 different transgenic lines was digested with Kpnl and XbaJ to release the 2.0 kb CGS cDNA insert and subjected to Southern blot analysis with 32 P-labeled Arabidopsis CGS DNA. The results showed that all transgenic lines contained the 2.0 kb
  • Kpnl-X bal CGS cDNA fragment, indicating that all lines are transformed.
  • the Arabidopsis probe did not pick up any signal in the DNA from the wild-type potato plants.
  • Southern blot analysis with genomic DNA digested with KpnX revealed that five transgenic lines are independent transformants. These transgenic lines, CGSl, 2, 4, 8, 10 were chosen for further analysis.
  • CGS activity was determined in the leaves and roots of transgenic plants and compared to the activity in the leaves and roots of wild-type potato plants.
  • the results shown in Table 1 indicate that CGS enzymatic activity is elevated in the leaves and roots of all transgenic potato lines.
  • five CGS[+] lines, CGSl, 2, 4, 8, and 10 had 2- to 7-fold higher CGS activity in their leaves, with CGS 1 and 8 as the highest.
  • CGS activity in the roots of transgenic lines was 2- to 4-fold higher than the CGS activity in the roots of wild-type plants.
  • Table 1 CGS enzyme activity in transgenic potato lines over-expressing CGS
  • CGS enzyme activity was measured in the leaf and root of potato plants. The averages ⁇ SE of two independent experiments are shown.
  • Free methionine levels in each transgenic potato line was determined by HPLC analysis using ethanol extraction.
  • the results shown in Table 2 and Fig. 2 indicate that the methionine levels in the leaves of transgenic potato plants were similar to the levels in the wild-type plants.
  • the levels of other amino acids analyzed, such as Thr and Tyr were similar to the levels in the leaves of wild-type plants.
  • Met levels in the tubers of transgenic plants were 2- to 5-fold higher than the levels in wild-type tubers, with CGSl as the highest.
  • Met levels in the roots of transgenic potato plants were 2- to 2.5-fold higher than the wild-type roots.
  • SAMS[+] construct transgenic Arabidopsis transformed with pO35SSAM (SAMS[+] construct) has been previously described (de Carvalho, et al, "Post- transcriptional gene silencing in transgenic plants," in Plant Molecular Biology: Molecular Genetic Analysis of plant development and metabolism, (NATO-ASI Series H, Vol. 81), Coruzzi et al, eds., Berlin, Springer Verlag (1994) on pages 437-452)). SAMS[+] plants were raised in a 23 °C growth chamber with a light intensity of ⁇ 100 ⁇ E/m/s and a photoperiod of 14 hr. light followed 10 hr. of darkness.
  • Plant lines derived from transformation with the SAMS[+] construct produced progeny with widely differing morphologies resulting from co-suppression of SAMS classified MUT1, MUT2, and MUT3 by severity of phenotypes.
  • MUTl overexpressed SAMS.
  • Stunted plants termed MUT2 and MUT3
  • All the plants appeared normal during the early stages of growth but as they grew the morphological types appeared when a switch to the co- suppressed state occurs, and this state persisted throughout further development (de Carvalho, et al, supra).
  • MUT3 became co-suppressed the earliest while MUT2 became co-suppressed late.
  • MUT3 plants were strikingly similar to those of CGS[-] plants (photographs not shown) in that numerous apical shoots were produced that failed to develop fully and the oldest leaves become thickened and curled (photograph not shown). Another similarity is that MUT3 plants were unable to flower. Although MUT2 plants were able to flower, the inflorescences were stunted and distorted (photographs not shown). SAMS suppressed plants differ from CGS[-] (photographs not shown) in that Met feeding had no effect on the ability of MUT3 to flower as it did on the CGS[-] plants. Although the frequencies with which the SAMS plant-types were produced is unpredictable, drought or antibiotic stress during the early growth period were noted to promote the formation of MUT2 and MUT3 and decrease the frequency with which the MUTl type was produced.
  • the present invention pertains to the field of agricultural biotechnology and food science, and entails the production of transgenic plants that when processed into various food products, possess enhanced flavor quality and/or stability. It also pertains to methods for selecting transformed plant cells using a specific selection agent and marker gene combination. The method is useful in selecting plant cells that express a nucleic acid of interest versus plant cells that do not express the nucleic acid of interest.

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Abstract

L'invention concerne des plantes transgéniques pourvues de parties comestibles qui produisent de la méthionine pendant le traitement. Les plantes contiennent des niveaux de méthionine élevés de telle manière que suite au traitement des parties comestibles, les niveaux de méthionine sont augmentés et occasionnent des produits alimentaires possédant une stabilité et/ou une qualité de saveur accrue. Les plantes préférées de cette invention sont les plantes de la famille Solanaceous, par exemple, la pomme de terre, la tomate, et l'aubergine, et d'autres plantes productrices de méthionine, dont le maïs et le soja. L'invention concerne également plusieurs manières d'organiser génétiquement des plantes pour produire des niveaux élevés exempts de Met, en introduisant un acide nucléique non-natif codant la synthétase de cystathionine gamma (CGS) et l'expression spécifique de tissu d'une synthétase de S-adénosyl méthionine anti-sens. Cette invention a également trait à des méthodes de sélection de cellules de plantes transformées au moyen d'éthionine et de CGS, respectivement en tant qu'agent de sélection et gène marqueur.
PCT/US2000/007330 1999-03-18 2000-03-20 Plantes transgeniques possedant des proprietes de saveur accruees WO2000055303A2 (fr)

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US6608239B1 (en) 1998-07-07 2003-08-19 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Means and methods for enhancing the content of sulfur compounds in plants
WO2005098015A3 (fr) * 2004-04-06 2006-07-20 Metanomics Gmbh Procede de production de produits chimiques fins
EP2390256A1 (fr) 2001-05-30 2011-11-30 Agrisoma, Inc. Chromosomes artificiels de plantes, leurs utilisations et leurs procédés de préparation
WO2013030812A1 (fr) * 2011-09-04 2013-03-07 Gavish-Galilee Bio Applications Ltd. Semences transgéniques de soja riche en méthionine exprimant le gène de la cystathionine gamma-lyase de l'arabidopsis
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6608239B1 (en) 1998-07-07 2003-08-19 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Means and methods for enhancing the content of sulfur compounds in plants
WO2001075130A1 (fr) * 2000-04-04 2001-10-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Plantes transgeniques a teneur accrue en methionine
EP2390256A1 (fr) 2001-05-30 2011-11-30 Agrisoma, Inc. Chromosomes artificiels de plantes, leurs utilisations et leurs procédés de préparation
WO2005098015A3 (fr) * 2004-04-06 2006-07-20 Metanomics Gmbh Procede de production de produits chimiques fins
US7951565B2 (en) 2004-04-06 2011-05-31 Metanomics Gmbh Process for the production of fine chemicals
US20150106971A1 (en) * 2011-09-02 2015-04-16 Philip Morris Products S.A. Threonine synthase from nicotiana tabacum and methods and uses thereof
US10501732B2 (en) 2011-09-02 2019-12-10 Philip Morris Products S.A. Threonine synthase from nicotiana tabacum and methods and uses thereof
WO2013030812A1 (fr) * 2011-09-04 2013-03-07 Gavish-Galilee Bio Applications Ltd. Semences transgéniques de soja riche en méthionine exprimant le gène de la cystathionine gamma-lyase de l'arabidopsis

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