EP0925366A1 - Verfahren zur herstellung von carotenoiden und speziellen ölen in pflanzensamen - Google Patents

Verfahren zur herstellung von carotenoiden und speziellen ölen in pflanzensamen

Info

Publication number
EP0925366A1
EP0925366A1 EP97938203A EP97938203A EP0925366A1 EP 0925366 A1 EP0925366 A1 EP 0925366A1 EP 97938203 A EP97938203 A EP 97938203A EP 97938203 A EP97938203 A EP 97938203A EP 0925366 A1 EP0925366 A1 EP 0925366A1
Authority
EP
European Patent Office
Prior art keywords
gene
plant
seed
carotenoid
carotenoid biosynthesis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97938203A
Other languages
English (en)
French (fr)
Inventor
Christine K. Shewmaker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monsanto Co
Original Assignee
Calgene LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Calgene LLC filed Critical Calgene LLC
Publication of EP0925366A1 publication Critical patent/EP0925366A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/8242—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
    • C12N15/8247—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 involving modified lipid metabolism, e.g. seed oil composition
    • 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
    • C12N15/825—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 involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

Definitions

  • the invention relates to genetic modification of plants, plant cells and seeds, particularly altering carotenoid biosynthesis and fatty acid composition.
  • Carotenoids are pigments with a variety of applications. They are yellow- orange-red lipids which are present in green plants, some molds, yeast and bacteria. Carotenoid hydrocarbons are referred to as carotenes, whereas oxygenated derivatives are referred to as xanthophylls.
  • the carotenoids are part of the larger isoprenoid biosynthesis pathway which, in addition to carotenoids, produces such compounds as chlorophyll and tocopherols, Vitamin E active agents.
  • the carotenoid pathway in plants produces carotenes, such as ⁇ - and ⁇ -carotene, and lycopene, and xanthophylls, such as lutein.
  • the biosynthesis of carotenoids involves the condensation of two molecules of the C 20 precursor geranyl PP ; to yield the first C 0 hydrocarbon phytoene.
  • phytoene yields lycopene.
  • Lycopene is the precursor of the cyclic carotenes, ⁇ -carotene and ⁇ -carotene.
  • the xanthophylls, zeaxanthin and lutein are formed by hydroxylation of ⁇ -carotene and ⁇ -carotene, respectively.
  • ⁇ -carotene a carotene whose color is in the spectrum ranging from yellow to orange, is present in a large amount in the roots of carrots and in green leaves of plants, ⁇ -carotene is useful as a coloring material and also as a precursor of vitamin A in mammals.
  • Current methods for commercial production of ⁇ -carotene include isolation from carrots, chemical synthesis, and microbial production.
  • a number of crop plants and a single oilseed crop are known to have substantial levels of carotenoids, and consumption of uch natural sources of carotenoids have been indicated as providing various beneficial health effects.
  • the below table provides levels of carotenoids that have been reported for various plant species.
  • Application WO 96/13149 reports on enhancing carotenoid accumulation in storage organs such as tubers and roots of genetically engineered plants.
  • the application is directed towards enhancing colored native carotenoid production in specific, predetermined non-photosynthetic storage organs.
  • the examples of the application are drawn to increasing colored carotenoids in transformed carrot roots and in orange flesh potato tubers. Both of these tissues are vegetative tissues, not seeds, and natively have a high level of carotenoids.
  • Carotenoids are useful in a variety of applications.
  • carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredents, as feed additives in aminal feeds and as colorants.
  • phytoene finds use in treating skin disorders. See, for example, U.S. Patent No. 4,642,318. Lycopene, ⁇ - and ⁇ -carotene are used as food coloring agents.
  • Plant oils are useful in a variety of industrial and edible applications. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources are needed. Depending upon the intended oil use, various different fatty acid compositions are desired. The demand for modified oils with specific fatty acid compositions is great, particularly for oils high in oleic acid. See, Haumann, B. F. (1996) INFORM 7:320-334. As reported by Haumann, the ideal frying oil would be a low-saturate, high oleic and low linolenic oil. Furthermore, studies in recent years have established the value of monoun aturated fatty acids as a dietary constituent.
  • oxidative stability of vegetable oil is related to the number of double bonds in its fatty acids. That is, molecules with several double bonds are recognized to be more unstable.
  • scientists have attempted to reduce the content of ⁇ -linolenic acid in order to improve shelf life and oxidative stability, particularly under heat.
  • Transformed plants, plant cells and seeds having altered carotenoid levels and/or modified fatty acid compositions are provided.
  • the plants, plant cells and seeds are transformed with at least one carotenoid biosynthesis gene.
  • Methods for making and using the transformed compositions of the invention are also provided. Methods find use in altering carotenoid levels in plants, particularly seeds, as well as increasing particular compounds for molecular farming, such as for production of particular carotenoids and tocopherols.
  • the transformed compositions, particularly seeds provide a source of modified oils, which oils may be extracted from the seeds in order to provide an oil product comprising a natural source of various carotenoids and carotenoid mixtures.
  • transformed seed can provide a source for particular carotenoid compounds and/or for modified speciality oils having altered carotenoid or tocopherol compostions and/or altered fatty acid composition, particularly having increased levels of oleic acid and decreased levels of linoleic and linolenic acids.
  • Figure 1 shows the nucleotide sequence of the SSU/crtB fusion sequence.
  • Figure 2 presents constructs for expression of carotenoid biosynthesis genes in plant seeds.
  • Figure 2A shows plasmid pCGN3390 which contains the napin promoter operably linked to the SSU/crtB sequence.
  • Figure 2B shows plasmid pCGN3392which contains the napin promoter operably linked to the SSV/crtE sequence.
  • Figure 2C shows plasmid pCGN9010 which contains the napin promoter operably linked to the SSU/crt/ sequence.
  • Figure 2D shows plasmid pCGN9009 which contains the napin promoter operably linked to the SSU/crtB sequence and the napin promoter operably linked to the SSU/crt7 sequence.
  • Figure 2E shows plasmid pCGN9002 which contains the napin promoter operably linked to the SSU/o _3 sequence and the napin promoter operably linked to an antisense epsilon cyclase sequence.
  • Figure 2F shows plasmid pCGN9017 which contains the napin promoter operably linked to the SSU/crt_3 sequence and the napin promoter operably linked to an antisense beta cyclase sequence.
  • Figure 3 shows the results of analyses of saponified samples for control seeds.
  • Figure 4 shows the results of analyses of saponified samples for pCGN3390 transformed seeds.
  • Figure 5 shows a graph of the fatty acid analysis in pCGN3390 transformed seeds and demonstrates that the increase in 18: 1 fatty acids correlates with a decrease in 18:2 and 18:3.
  • Figure 6 shows a graph of the fatty acid analysis in pCGN3390 transformed seeds and demonstrates that the increase in 1 : 1 correlates with an increase in both 18:0 and 20:0, but little effect is seen in 16:0.
  • Figure 7 shows a graph of the fatty acid analysis in pCGN3390 transformed seeds and demonstrates the increase in 18:0 correlates well with an increase in 20:0.
  • Figure 8 shows a carotenoid biosynthesis pathway.
  • Figure 9 provides sequence of 5. napus epsilon cyclase cDNA clone 9-4.
  • FigurelO provides sequence of B. napus epsilon cyclase cDNA clone 7-6.
  • Figure 11 provides sequence of a B. napus beta cyclase cDNA clone.
  • Figure 12 provides T2 seed analysis of 3390 transformed Brassica napus plants.
  • Figure 13 provides T3 seed analysis of 3390 transformed Brassica napus plants.
  • methods for increasing production of carotenoid compounds as well as for altering fatty acid compositions in a plant, particularly in plant seeds are provided.
  • the method involves transforming a plant cell with at least one carotenoid biosynthesis gene. This has the effect of altering carotenoid biosynthesis particularly increasing the production of downstream products, as well as providing novel seed oils having desirable fatty acid compositions.
  • a second gene can then be utilized to shunt the metabolic activity to the production of particular carotenoid compounds or to further alter the fatty acid composition.
  • transfonnation of a plant with an early carotenoid biosynthesis gene leads to a significant increase in the flux through the carotenoid pathway resulting in an increase in particular carotenoids.
  • the transformed seeds may demonstrate altered fatty acid compositions as the result of the carotenoid gene expression, such as seen with the seeds described herein from plants transformed with a phytoene synthase gene.
  • oilseed Brassica for example, transformation with an early carotenoid biosynthesis gene leads to seeds having a significant increase in the production of ⁇ -carotene, ⁇ -carotene and lutein.
  • the Brassica seeds demonstrate an altered fatty acid composition and yield a vegetable oil which has increased oleic acid content and decreased linoleic and linolenic acid content.
  • the transformed seed can provide a source of carotenoid products as well as modified seed oil. In this manner, modified speciality oils can be produced and new sources of carotenoids for extraction and purification are provided.
  • the oils of the present invention also provide a substantial improvement with respect to stability as compared to two other major plant sources of carotenoids, marigold petals and red palm oil (mesocarp). Although instability is observed in seeds stored in air at room temperature as demonstrated by loss of approximately 20-30% of total carotenoids after 4 weeks of storage, the loss after 1-2 weeks is only 10%. Palm mesocarp, by contrast, must be processed within a day or two of harvest in order to avoid major losses of carotenoid. Furthermore, the carotenoid decomposition in the seeds of the present invention may be reduced significandy by storage of the seeds under nitrogen.
  • early carotenoid biosynthesis gene For the production of a seed having an increase in carotenoid biosynthesis, transformation of the plant with an early carotenoid biosynthesis gene is sufficient.
  • early carotenoid biosynthesis gene is intended geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and isopentenyl diphosphate (IPP) isomerase.
  • IPP isopentenyl diphosphate
  • IPP isomerase has been isolated from: 7?. Capsulatus (Hahn et al. (1996) ./. Bacterial. 178:61 -624 and the references cited therein), GenBank Accession Nos.
  • Transformation with an early carotenoid gene increases the biosynthetic activity of the carotenoid pathway, and can lead to increased production of particular carotenoids such as for example, ⁇ - and ⁇ -carotene.
  • carotenoids such as for example, ⁇ - and ⁇ -carotene.
  • a phytoene synthase as the primary gene, large increases in the carotenoid content generally, and particularly in the levels of ⁇ - and ⁇ -carotene, are obtained in seeds of transformed plants.
  • Oil comprising the carotenoids so produced may be extracted from the seeds to provide a valuable source of ⁇ - and ⁇ -carotenes.
  • Such an oil may find use as a food colorant, for example to add color to margarines, or as a food oil.
  • An edible food oil with high ⁇ - and ⁇ -carotene levels is of interest for prevention of Vitamin A deficiency which can result in night blindness.
  • production of the transformed plants and extraction of the high ⁇ - and ⁇ -carotene oil to provide a useful food oil is particularly desirable in regions where night blindness is a widespread problem, such as in India and Asia.
  • levels of other carotenoids are also increased in the oils exemplified herein.
  • lutein levels are increased in seeds from plants transformed with a phytoene synthase gene, as well as in seeds from plants transformed with a GGPP synthase gene.
  • additional primary genes may be expressed to provide for even greater flux through the carotenoid pathway.
  • increased levels of phytoene are observed in the oilseed Brassica seeds containing the phytoene synthase gene as described herein.
  • increasing the expression of phytoene desaturase as well as the phytoene synthase may result in further increases in the levels of carotenoids, such as ⁇ - and ⁇ -carotene and lutein, produced.
  • plants expressing both the phytoene synthase and the GGPP synthase genes are desirable and may be produced by crossing the 3390 and 3392 plants comprising these genes which are described herein.
  • the pathway can be diverted for the production of specific compounds.
  • the diversion involves the action of at least one second gene of interest, (the secondary gene).
  • the secondary gene can encode an enzyme to force the production of a particular compound or alternatively can encode a gene to stop the pathway for the accumulation of a particular compound.
  • expression of a carotenoid biosynthesis gene in the pathway for the desired carotenoid compound is used.
  • Genes native or foreign to the target plant host may find use in such methods, including, for example carotenoid biosynthesis genes from sources other than higher plant, such as bacteria, including Erwinia and
  • Rhodobacter species For stopping the pathway in order to accumulate a particular carotenoid compound, the secondary gene will provide for inhibition of transcription of a gene native to the target host plant, wherein the enzyme encoded by the inhibited gene is capable of modifying the desired carotenoid compound. Inhibition may be achieved by transcription of the native gene to be inhibited in either the sense
  • ⁇ -carotene derived carotenoids such as zeaxanthin, zeaxanthin diglucoside, canthaxanthin, and astaxanthin
  • inhibition of lycopene epsilon cyclase is desired to prevent accumulation of alpha carotene and it's derivative carotenoids, such as lutein.
  • increased expression of a secondary gene may be desired for increased accumulation of a particular beta-carotene derived carotenoid.
  • increased ⁇ -carotene hydroxylase expression is useful for production of zeaxanthin
  • e increased ⁇ -carotene hydroxylase and keto-introducing enzyme expression is useful for production of astaxanthin.
  • inhibition of lycopene beta cyclase or of lycopene epsilon cyclase and lycopene beta cyclase is desired to reduce conversion of lycopene to alpha- and beta-carotene.
  • ⁇ -carotene hydroxylase or crtZ (Hundle et al. (1993) FEBS Lett. 375:329-334, GenBank Accession No. M87280) for the production of zeaxanthin; genes encoding keto-introducing enzymes, such ascrtW (Misawa et al. (1995) ./. Bacteriol. 777:6575-6584, WO 95/18220, WO 96/0 172) or ⁇ -C-4-oxygenzse (crtO; Harker and Hirschberg (1997) FEBS Lett.
  • the pathway can also be manipulated to decrease levels of a particular carotenoid by transformation of antisense DNA sequences which prevent the conversion of the presursor compound into the particular carotenoid being regulated.
  • Secondary genes can also be selected to alter the fatty acid content of the plant for the production of speciahty oils.
  • acyl-ACP thioesterase genes having specificity for particular fatty acid chain lengths may be used. See, for example, USPN 5,304,481, USPN 5,455,167, WO 95/13390, WO 94/10288, WO 92/20236, WO 91/16421 , WO 97/12047 and WO 96/36719.
  • fatty acid biosynthesis genes of interest include, but are not limited to, ⁇ -keto acyl-ACP synthases (USPN 5,510,255), fatty acyl CoA synthases (USPN 5,455,947), fatty acyl reductases (USPN 5,370,996) and stearoyl-ACP desaturases (WO 91/13972).
  • a high stearate content may be obtained in seeds by expression of a mangosteen acyl-ACP thioesterase.
  • a mangosteen acyl-ACP thioesterase To combine the high oleic acid trait of the 3390 plants described herein with the 5266 high stearate plants described in WO 97/12047, crosses were made between 3390-1 and 5266-35 and between 3390-1 and 5266-5. Seeds resulting from these crosses contained oil having a high stearate, low linoleic, low linolenic and high carotenoid phenotype.
  • any means for producing a plant comprising the primary gene or both the primary and secondary genes are encompassed by the present invention.
  • the secondary gene of interest can be used to transform a plant at the same time as the primary gene (cotransformation), the secondary gene can be introduced into a plant which has already been transformed with the primary gene, or alternatively, transformed plants, one expressing the primary gene and one expressing the secondary gene, can be crossed to bring the genes together in the same plant.
  • the carotenoid levels can be altered in particular tissues of the plant.
  • carotenoid levels in the seed including embryos and endosperm, can be altered by the use of seed specific transcriptional initiation regions. Such regions are disclosed, for example, in U.S.
  • Patent No. 5,420,034 which disclosure is herein incorporated by reference.
  • the transformed seed provides a factory for the production of modified oils.
  • the modified oil may be used or alternatively, the compounds in the oils can be isolated.
  • the present invention allows for the production of particular compounds of interest as well as speciality oils.
  • the primary or secondary genes encoding the enzymes of interest can be used in expression cassettes for expression in the transformed plant tissues.
  • the plant is transformed with at least one expression cassette comprising a transcriptional initiation region linked to a gene of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the transcriptional initiation may be native or analogous to the host or foreign or heterologous to the host. By foreign is intended that the transcriptional initiation region is not found the wild-type host into which the transcriptional initiation region is introduced.
  • acyl carrier protein ACP
  • the transcriptional cassette will include the in 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the tennination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., (1991), Mol. Gen.
  • the expression cassette will additionally contain a gene encoding a transit peptide to direct the gene of interest to the plastid.
  • transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104- 126; Clark et al. (1989) ./. Biol. Chem. 264: 17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer etal. ( ⁇ 993) Biochem. Biophys. Res Commun. 796:1414-1421; and. Shah et al. (1986) Science 233:478-481. Plant carotenoid genes useful in the invention may utilize native or heterologous transit peptides. It is noted that where the gene or DNA sequence of interest is an antisense
  • DNA, targeting to a plastid is not required.
  • the construct may also include any other necessary regulators such as plant translational consensus sequences (Joshi, C.P., (1 87), Nucleic Acids Research, 75:6643-6653), introns (Luehrsen and Walbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operably linked to the nucleotide sequence of interest.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 754:9-20), and human immunoglobulin heavy-chain binding protein
  • the sequence may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons.
  • the plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17: 477-498.
  • the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
  • chloroplast preferred genes see USP ⁇ 5,545,817.
  • the various D ⁇ A fragments may be manipulated, so as to provide for the D ⁇ A sequences in the proper orientation and, as appropriate in the proper reading frame.
  • adapters or linkers may be employed to join the D ⁇ A fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous D ⁇ A, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resection, hgation, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and transversions, may be involved.
  • the reeombinant DNA molecules of the invention can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) BioTechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrohacterium mediated transformation (Hinchee et al.
  • a plant plastid can be transformed directly. Stable transformation of chloroplasts has been reported in higher plants, see, for example, SVAB et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:8526-8530; SVAB & Maliga (1993) Proc. Nat'l Acad. Sci. USA 90:913-917; Staub & Maliga (1993) Embo J. 12:601-606.
  • the method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination.
  • plastid gene expression can be accomplished by use of a plastid gene promoter or by trans-activation of a silent plastid-borne transgene positioned for expression from a selective promoter sequence such as that recognized by T7 RNA polymerase.
  • the s ⁇ ent plastid gene is activated by expression of the specific RNA polymerase from a nuclear expression construct and targeting of the polymerase to the plastid by use of a transit peptide.
  • Tissue-specific expression may be obtained in such a method by use of a nuclear-encoded and plastid-directed specific RNA polymerase expressed from a suitable plant tissue specific promoter.
  • the cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al.. Plant Cell Reports
  • any plant variety may be employed.
  • plant species which provide seeds of interest.
  • plants will be chosen where the seed is produced in high amounts, a seed-specific product of interest is involved, or the seed or a seed part is edible.
  • Seeds of interest include the oil seeds, such as oilseed Brassica seeds, cotton seeds, soybean, safflower, sunflower, coconut, palm, and the like; grain seeds, e.g.
  • seed transcriptional initiation regions are used in combination with at least one carotenoid biosynthesis gene. This increases the activity of the carotenoid pathway and alters carotenoid levels in the transformed seed. In this manner, particular genes can be selected to promote the formation of compounds of interest.
  • the transformed seed has a significant increase in carotenoid biosynthesis as the result of an icrease in the flux through the pathway.
  • the gene selected is an early carotenoid biosynthesis gene
  • Brassica seeds transformed with an early carotenoid biosynthesis gene significant increases in the production of ⁇ -carotene, ⁇ - carotene and smaller increases in lutein in the seed oil, as well as altered oil fatty acid compositions are obtained.
  • significant increases of a particular carotenoid include those ranging from a 10 to a 50 fold increase, preferably at least a 50 to a 100 fold increase, more preferably, at least a 50 to a 200 fold increase, such as the increases seen in ⁇ -carotene and ⁇ -carotene levels. Lutein levels, in this case, are also increased, but lower increases of 1.5 - 2 fold are obtained. At the same time, total carotenoid levels may be increased at least 10 to 25 fold, preferably 25 to 60 fold, and more preferably 25 to 100 fold.
  • a seed of the invention transformed with a phytoene synthase gene has a substantial increase in levels of ⁇ - and ⁇ -carotene and total carotenoids, as well as smaller increases in lutein and other carotenoids. In some cases, it is not possible to quantitate the fold increase in a given carotenoid compound, as the levels are too low to detect in seeds from non- transfonned.
  • ⁇ -cryptoxanthin, lycopene, phytoene and phytofluene are all detected in various levels in seeds transformed with a crtB gene, but are not detectable in seeds from untransformed Brassica napus plants.
  • the early carotenoid biosynthesis gene is GGPP synthase
  • 1.5 to 2 fold increases in lutein and ⁇ -carotene may be obtained.
  • Lycopene is also detected in seeds from Brassica napus plants transformed with a crtE (GGPP synthase) gene.
  • Total carotenoids in this case are also increased approximately 2 fold.
  • plants which have been engineered to express increased levels of both phytoene synthase and GGPP synthase are plants which have been engineered to express increased levels of both phytoene synthase and GGPP synthase.
  • This metabolic energy effected by transformation with an early carotenoid gene can be funneled into a metabolic compound of choice by transformation with a second gene.
  • the second gene is designed to promote the synthesis of a particular carotenoid by promoting the formation of the carotenoid of interest or alternatively by stopping the pathway to allow for the buildup of compounds.
  • the seeds of the invention which have been transformed with the primary early carotenoid biosynthesis gene also provide a source for novel oil compositions.
  • the use of phytoene synthase as the primary gene results in substantial increases in oleic acid content in seed oil.
  • substantial increase is intended an increase of from about 5% to about 40%, specifically from about 20% to about 40%, more specifically from about 30% to about 40%.
  • the seeds of the invention which have been transformed with a primary early carotenoid biosynthesis gene provide a source for modified oils having a high oleic acid content. That is, carotenoid biosynthesis genes, particularly early carotenoid biosynthesis genes can be used to produce seeds having at least 70% oleic acid, on a weight percentage basis.
  • the oleic acid content in any seed can be altered by the present methods, even those seeds having a naturally high oleic acid content. Alteration of seeds having naturally high oleic acid contents by the present methods can result in total oleic acid contents of as high as 80%.
  • linoleic and linolenic acid content there is also a decrease in linoleic and linolenic acid content.
  • decrease in linoleic fatty acid content is intended a decrease from about 10% to about 25%, preferably about 25% to about 40%, more preferably about 35% to about 60%.
  • decrease in linolenic fatty acid content is intended a decrease from about 10% to about 30%, preferably about 30% to about 60%, more preferably about 50% to about
  • the methods of the invention result in oils which are more oxidatively stable than the naturally occurring oils.
  • the modified oils of the invention are low- saturate, high oleic and low linolenic.
  • the present invention provides oils high in monounsaturated fatty acids which are important as a dietary constituent.
  • seed oil can be modified to engineer an oil with a high oleic acid content as well as a high level of a carotenoid of interest.
  • High oleic acid and and high ⁇ - and ⁇ -carotene oils would have a longer shelf life as both the oleic acid and ⁇ - and ⁇ -earotene content would lend stability. It is also noted that such oils are more desirable as sources of carotenoids than the natural red palm oil, which oil contains high levels of saturated fatty acids.
  • the transformed seed of the invention can thus provide a source of carotenoid products as well as modified fatty acids.
  • methods are available in the art for the purification of the carotenoid compounds.
  • methods available in the art can be utilized to produce oils purified of carotenoids. See, generally, WO 96/13149 and Favati et al. (1988) J. Food Sci. 53:1532 and the references cited therein.
  • the tocopherol levels can be altered, preferably increased. Such seeds with increased levels of tocopherol, particularly ⁇ -tocopherol, are desirable as ⁇ -tocopherol is the most important form of the vitamin E family.
  • Vitamin E is essential for the nutrition of humans and other animals. Evidence is available that vitamin E functions in the body in maintaining the integrity of the red blood cells, as essential in cellular respiration, is involved in the biosynthesis of DNA, and acts as an antioxidant which may have implications in protecting cells from carcinogens. Thus, seeds and oils having increased tocopherol levels are desirable. Oils having a nearly 50% increase in ⁇ -tocopherol levels are provided herein, and seed oils having even greater increases, up to 2-5 fold, are envisioned using the methods of the present invention.
  • the transformed seed and embryos additionally find use as screenable markers. That is, transformed seed and embryos can be visually determined and selected based on color as a result of the increased carotenoid content.
  • the transformed seeds or embryos display a color ranging from yellow to orange to red as a result of the increased carotenoid levels. Therefore, where plant transformation methods involve an embryonic stage, such as in transformation of cotton or soybean, the carotenoid gene can be used in plant transformation experiments as a marker gene to allow for visual selection of transformants. Likewise, segregating seed can easily be identified as described further in the following examples.
  • SSU fusions to E. uredovora carotenoid biosynthesis genes (1 ) Phytoene Synthase
  • the SSU leader and crtB gene sequences were joined by PCR.
  • the sequence of the SSU/crtB fusion is shown in Figure 1.
  • the crtB gene from nucleotides 5057 to 5363 (numbering according to Misawa et al. (1990) supra) was joined to the SSU leader as follows.
  • a Bgl site was included upstream of the SSU leader start site to facilitate cloning.
  • the thymidine nucleotide at 5057 of crtB was changed to an adenosine to make the first amino acid at the SSU leader/crt ⁇ junction a methionine, and the splice site a cys-met-asn.
  • the native splice site for SSU is csy-met-gln.
  • Misawa et al. (1990) supra) indicates that the start site for the coding region for crtB is at nucleotide 5096.
  • a plasmid comprising a E. uredovora crtl gene fused to the transit peptide sequence of the pea Rubisco small subunit was described by Misawa et al. (The Plant Journal (1993) 4:833-840.
  • An approximately 2.1 kb XbaVEcoRl fragment of this plasmid containing the SSU-crt/ fusion and a nos 3' termination region was cloned in position for expression from a napin 5' promoter.
  • pCGN1559PASS is a binary vector for _4#r ⁇ b ⁇ cter.Mm-mediated transformation such as those described by McBride et al. (Plant Mol. Biol. (1990) 74:269-276) and is prepared from pCGN1559 by substitution of the pCGN1559 tinker region with a linker region containing the following restriction digestion sites: Bam jSwa ⁇ ]SseK3Xl(Pst ⁇ )/Hin ⁇ m.
  • a map of pCGN3390 is provided in Figure 2A.
  • a fragment comprising a napin 5'/SSU-c ⁇ t7 fusion/nos 3' construct as described above was cloned into a binary vector for plant transformation resulting in pCGN9010.
  • a map of pCGN9010 is provided in Figure 2C.
  • (3) GGPP Synthase pCGN3360 carrying the complete SSU/crtE fusion was cut with BgH ⁇ and BamH to excise the SSU/crtE fusion.
  • the resulting 1.2 kb fragment was ligated into the napin expression cassette in pCGN3223 at the BamHI site.
  • pCGN3391 was digested with H dEQ to excise the napin promoter-SSU/crtE napin 3' fragment, which was then cloned into HmdIII cut pCGN1559PASS yielding pCGN3392.
  • a map of pCGN3392 is provided in Figure 2B.
  • BrUxSsica napus epsilon cyclase genes are isolated by PCR using primers designed from an Arabidopsis epsilon cyclase gene (Cunningham FX Jr (1996) Plant Cell 8: 1613-1626). Sequence of B. napus epsilon cyclase genes is provided in Figures
  • An antisense construct is prepared by cloning anXhol/BamHl fragment of cDNA clone 9-4 into a napin expression cassette (pCGN3223) digested with Xhol and Bg ⁇ .
  • the napin 5'-antisense epsilon cyclase- napin 3' fragment is cloned along with a napin 5'-SSU/crt#-napin 3' fragment, fragment into a binary vector for plant transfo ⁇ nation, resulting in pCGN9002, shown in Figure 2E.
  • Brassica napus beta cyclase genes are isolated by PCR using primers designed from an Arabidopsis beta cyclase gene (Cunningham FX Jr (1996) Plant Cell 8:1613- 1626). Sequence of a _5. napus beta cyclase cDNA, 32-3, is provided in Figures 1 1.
  • An antisense construct is prepared by cloning anXho I fragment of the beta cyclase cDNA clone into a napin expression cassette (pCGN3223) digested with Xhol. A clone containing the beta cyclase in the antisense orientation is selected. The napin 5 '- antisense beta cyclase-napin 3' fragment is cloned along with a napin 5'-SSU/crt_5- napin 3' fragment into a binary vector for plant transformation, resulting in pCGN9017, shown in Figure 2F.
  • the napin-SSU leader/crt ⁇ plants in 212/86 were tagged at 21 days, 28 days and 35 days post anthesis.
  • 3390-1 was harvested at 28 days, some of the seeds were obviously orange. AT 35dpa, the orange was obvious enough that a segregation ratio could be obtained. This trend of orange seeds has continued and is seen in each of the 17 tines harvested that have been obtained.
  • a table of the segregation ratios is included below in Table 1.
  • Carotenoids were extracted from seeds harvested at approximately 35 days post-anthesis as follows. Eight seed samples of orange seeds from transgenic plant 3390-1 and eight seed samples of a 212/86 variety rapeseed control plant were ground in 200 ⁇ l of 70% acetone/30% methanol. The ground seed mixture was then spun in a microcentrifuge for approximately 5 minutes and the supernatant removed. Two additional 70% acetone/30% methanol extractions were conducted with the pelleted seed material and all three supernatants pooled and labeled A/M extract.
  • control seed pellets are white, whereas the seed pellets from the transgenic seeds have a yellow color.
  • the pellets are then extracted twice with ether and the resultant supernatants pooled and labeled E extract.
  • the A/M extract was then transferred to ether as follows. 450 ⁇ l ether and 600 ⁇ l of water were added to the extracts, followed by removal of the ether layers. The A/M extracts were then washed two more time with 400 ⁇ l of ether, and the ether fractions from the three A/M washes pooled. The E extracts described above were washed once with 400 ⁇ l of water and pooled with the A/M ether fractions. The pooled ether fractions were blown down to a volume of approximately 300 ⁇ l with nitrogen gas and filtered using a syringe microfilter.
  • the sample vials were rinsed with approximately lOO ⁇ l ether and the rinse was similarly filtered and pooled with the initial filtrate, yielding total volume of approximately 150 ⁇ l.
  • a 50 ⁇ l aliquot was stored at -20YC until further analysis and the remaining lOO ⁇ l sample was saponified as follows. lOO ⁇ l of 10% potassium hydroxide (KOH) in methanol was added to each l ⁇ O ⁇ l sample and the mixture stored in the dark at room temperature for approximately 2 hours. 400 ⁇ l of water was then added to the samples and the ether phase removed. For better phase separation, saturated NaCl may be substituted for the water. The water solution was then extracted twice more with lOO ⁇ l of ether and the ether samples pooled and washed with water.
  • KOH potassium hydroxide
  • the saponified samples were then analyzed by HPLC analysis on a Rainin microsorb C18 column (25cm length, 4.6mm outside diameter) at a flow rate of 1.5ml per minute.
  • the gradient used for elution is as follows:
  • the "Random" population includes a proportion of nulls.
  • the maroon population contains only transgenics. Due to an effort to exclude nulls from this population, the inclusion of homozygotes may be favored.
  • “other” includes mostly very polar compounds, such as neoxanthin, violaxanthin, etc.
  • transgenic sample “other” includes these and additional compounds, such as zeta-carotene, neurosporene, and mono-cyclic carotenoids.
  • Fatty Acid Analysis Fatty acid composition of mature seeds was determined by GC analysis of single T2 seeds harvested from trangenic plants 3390- 1 and 3390-8. Single seeds from both Random (R) and Maroon (M) populations (as defined above) were analyzed and compared to seeds from a 212/86 control (SP001-1). The results of these analyses are provided in Table 3 below as weight % total fatty acids.
  • Carotenoids were analyzed in mature T2 seeds of 3392 B. napus plants tranformed to express the E. uredovora crtE gene. An approximately two fold increase in levels of lutein and ⁇ -carotene was observed in seeds of plant 3392-SP30021-16. Lycopene was also detected in these seeds and is undetectable in seeds of untransf ormed control plants. Analysis of seeds from 7 additional 3392 transformants did not reveal significant increases in the carotenoid levels.
  • fatty acid composition can also be altered in the transgenic plant seeds.
  • seeds can be used to produce novel products, to provide for production of particular carotenoids, to provide high oleic oils, and the like.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nutrition Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Edible Oils And Fats (AREA)
EP97938203A 1996-08-09 1997-08-08 Verfahren zur herstellung von carotenoiden und speziellen ölen in pflanzensamen Withdrawn EP0925366A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2414596P 1996-08-09 1996-08-09
US24145P 1996-08-09
PCT/US1997/014035 WO1998006862A1 (en) 1996-08-09 1997-08-08 Methods for producing carotenoid compounds and speciality oils in plant seeds

Publications (1)

Publication Number Publication Date
EP0925366A1 true EP0925366A1 (de) 1999-06-30

Family

ID=21819082

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97938203A Withdrawn EP0925366A1 (de) 1996-08-09 1997-08-08 Verfahren zur herstellung von carotenoiden und speziellen ölen in pflanzensamen

Country Status (7)

Country Link
EP (1) EP0925366A1 (de)
JP (1) JP2001505409A (de)
CN (1) CN1227609A (de)
AU (1) AU4058497A (de)
BR (1) BR9713462A (de)
CA (1) CA2261577A1 (de)
WO (1) WO1998006862A1 (de)

Families Citing this family (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6642021B2 (en) * 1996-03-29 2003-11-04 University Of Maryland Methods of producing carotenoids by the expression of plant ε-cyclase genes
US6429356B1 (en) * 1996-08-09 2002-08-06 Calgene Llc Methods for producing carotenoid compounds, and specialty oils in plant seeds
US6653530B1 (en) 1998-02-13 2003-11-25 Calgene Llc Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US6600089B1 (en) 1998-04-24 2003-07-29 E. I. Du Pont De Nemours And Company Carotenoid biosynthesis enzymes
WO1999055887A2 (en) * 1998-04-24 1999-11-04 E.I. Du Pont De Nemours And Company Carotenoid biosynthesis enzymes
AU757440B2 (en) * 1998-08-05 2003-02-20 Sungene Gmbh And Co. Kgaa DNA sequence coding for a 1-deoxy-d-xylulose-5-phosphate synthase and the overproduction thereof in plants
WO2000011199A1 (en) * 1998-08-20 2000-03-02 Pioneer Hi-Bred International, Inc. COMPOSITIONS AND METHODS FOR ALTERING AN ACETYL-CoA METABOLIC PATHWAY OF A PLANT
CN1330718A (zh) * 1998-10-07 2002-01-09 辛根塔参与股份公司 植物中的治疗活性蛋白质
US6232530B1 (en) * 1998-11-30 2001-05-15 University Of Nevada Marigold DNA encoding beta-cyclase
US6515206B1 (en) * 1998-12-23 2003-02-04 Calgene Llc Plastid transformation of Brassica
WO2000049116A1 (en) * 1999-02-18 2000-08-24 Calgene Llc Method for refining a seed oil having micronutrients
DE19909637A1 (de) * 1999-03-05 2000-09-07 Peter Beyer Verfahren zur Verbesserung des agronomischen Wertes und der ernährungsphysiologischen Eigenschaften von Pflanzen
ATE446370T1 (de) * 1999-04-12 2009-11-15 Monsanto Technology Llc Öl, das brassicastanol enthält
JP2002541851A (ja) 1999-04-15 2002-12-10 カルジーン エルエルシー イソプレノイド合成に関与するタンパク質の核酸配列
SE9903336D0 (sv) * 1999-09-17 1999-09-17 Astacarotene Ab DNA construct and its use
AU2001247480A1 (en) * 2000-03-17 2001-10-03 Exelixis Plant Sciences, Inc. Melon promoters for expression of transgenes in plants
US6872815B1 (en) 2000-10-14 2005-03-29 Calgene Llc Nucleic acid sequences to proteins involved in tocopherol synthesis
AU2001261447A1 (en) * 2000-05-12 2001-11-26 Monsanto Technology Llc Method for producing carotenoid compounds in plants
CA2413793C (en) 2000-06-27 2013-01-08 Basf Aktiengesellschaft Cyanobacterial nucleic acid fragments encoding proteins useful for controlling plant traits via nuclear or plastome transformation
US7083967B1 (en) 2000-06-27 2006-08-01 Basf Corporation Cyanobacterial nucleic acid fragments encoding proteins useful for controlling plant traits via nuclear or plastome transformation
CA2418436C (en) 2000-08-07 2017-07-11 Monsanto Technology, Llc Methyl-d-erythritol phosphate pathway genes
ATE419366T1 (de) 2001-05-09 2009-01-15 Monsanto Technology Llc Tyra-gene und ihre verwendung
US7244877B2 (en) 2001-08-17 2007-07-17 Monsanto Technology Llc Methyltransferase from cotton and uses thereof
AR036962A1 (es) 2001-10-25 2004-10-13 Monsanto Technology Llc Metiltransferasas aromaticas y usos de las mismas
BRPI0308740B1 (pt) 2002-03-19 2018-11-21 Monsanto Technology Llc molécula de ácido nucléico codificando uma homogentisato prenil transferase (“hpt”) e método de produzir planta tendo semente com nível de tocoferol aumentado
EP1546334A4 (de) 2002-08-05 2007-01-03 Monsanto Technology Llc Gene in verbindung mit tocopherol-biosynthese und verwendungen davon
WO2004017749A2 (de) * 2002-08-20 2004-03-04 Sungene Gmbh & Co. Kgaa Verwendung von astaxanthinhaltigen pflanzen oder pflanzenteilen der gattung tagetes als futtermittel
JP2006508681A (ja) * 2002-12-06 2006-03-16 デル・モンテ・フレッシュ・プロデュース・カンパニー カロテノイドレベルを改変したトランスジェニックパイナップル植物体及びその作製方法
JP2006521107A (ja) * 2003-03-24 2006-09-21 シンジェンタ リミテッド 植物中のカロチノイド蓄積の増強
DE102004007624A1 (de) * 2004-02-17 2005-09-15 Sungene Gmbh & Co. Kgaa Neue Ketolasen und Verfahren zur Herstellung von Ketocarotinoiden
BRPI0615066A2 (pt) 2005-09-02 2011-05-03 Univ Cornell polinucleotìdeos que codificam enzimas do caminho biossintético de carotenóides e apocarotenóides em cafeeiro
CN100410376C (zh) * 2006-03-06 2008-08-13 中国科学院遗传与发育生物学研究所 与油脂代谢调控相关的转录因子GmDofC及其编码基因与应用
CA2678762A1 (en) * 2007-02-21 2008-08-28 Abdelali Hannoufa Altering carotenoid profiles in plants
ES2339094B1 (es) * 2008-11-13 2011-03-18 Iden Carbohydrate Biotechnology, S.L. Procedimiento para la produccion de plantas transgenicas que presentan alto contenido en compuestos antioxidantes, alta capacidad antioxidante y resistencia al empardecimiento.
CN101514345B (zh) * 2009-02-19 2012-07-04 上海交通大学 生菜植醇激酶蛋白编码序列
US20140123339A1 (en) 2012-10-31 2014-05-01 Pioneer Hi Bred International Inc Transformed Plants Having Increased Beta-Carotene Levels, Increased Half-Life and Bioavailability and Methods of Producing Such
CN104357479B (zh) * 2014-11-18 2017-06-06 西南大学 干扰黄色色素表达同时超量表达番茄红素在制备红色花瓣芸薹属植物中的应用
CN104480128B (zh) * 2014-11-18 2018-02-27 西南大学 抑制黄色色素积累同时积累番茄红素和花青素苷在制备花瓣呈红色芸薹属植物中的应用
CN104388464B (zh) * 2014-12-19 2017-04-26 西南大学 干扰lcyb、lcye表达并超量表达crtiso在制备红色花瓣芸薹属植物中的应用
CN104404079B (zh) * 2014-12-19 2017-03-15 西南大学 干扰lcyb、lcye表达同时超量表达gl3在制备花瓣呈红色芸薹属植物中的应用
CN104404080B (zh) * 2014-12-19 2017-06-23 西南大学 干扰lcyb、lcye表达同时超量表达egl3在制备花瓣呈红色芸薹属植物中的应用
CN104531755B (zh) * 2014-12-19 2017-06-06 西南大学 干扰lcyb、lcye表达同时异位表达tt8在制备红色花瓣芸薹属植物中的应用
CN104531754B (zh) * 2014-12-19 2017-05-31 西南大学 干扰lcyb、lcye表达同时超量表达pap在制备花瓣呈红色芸薹属植物中的应用
CN105695506A (zh) * 2016-04-20 2016-06-22 西南大学 一种改良棉籽营养品质的方法及其用途
EP3792353A1 (de) * 2019-09-13 2021-03-17 CRAG - Centre de Recerca en Agrigenomica CSIC-IRTA-UB-UAB Künstliche chromoplastenbiogenese
CN110656114B (zh) * 2019-10-18 2022-07-01 云南中烟工业有限责任公司 一种烟草色素合成相关的基因及其应用
CN111926036B (zh) * 2020-10-14 2020-12-29 中国农业科学院生物技术研究所 一种联合表达载体及其在玉米籽粒表达虾青素中的应用
CN113549639B (zh) * 2021-07-21 2022-07-29 云南中烟工业有限责任公司 一种降低烟叶总蛋白及烟气苯酚含量的调控基因

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4727219A (en) * 1986-11-28 1988-02-23 Agracetus Genic male-sterile maize using a linked marker gene
US5922602A (en) * 1988-02-26 1999-07-13 Biosource Technologies, Inc. Cytoplasmic inhibition of gene expression
AU6287390A (en) * 1989-08-01 1991-03-11 Pioneer Hi-Bred International, Inc. Transcriptional activators of anthocyanin biosynthesis as visual markers for plant transformation
US7705215B1 (en) * 1990-04-17 2010-04-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
GB8928179D0 (en) * 1989-12-13 1990-02-14 Ici Plc Dna,constructs,cells and plants derived therefrom
US5618988A (en) * 1990-03-02 1997-04-08 Amoco Corporation Enhanced carotenoid accumulation in storage organs of genetically engineered plants
CA2055447C (en) * 1990-03-02 2006-12-19 Rodney Lee Ausich Biosynthesis of carotenoids in genetically engineered hosts
RU2005352C1 (ru) * 1991-09-19 1994-01-15 Владимир Савельевич Феденко Способ определения специфичности эндоспермовых мутантов кукурузы к накоплению каротиноидов
GB9204583D0 (en) * 1992-03-03 1992-04-15 Unilever Plc Recombinant plant enzyme
WO1994011516A1 (en) * 1992-11-17 1994-05-26 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and related enzymes from plants
US7205457B1 (en) * 1993-02-05 2007-04-17 Monsanto Technology Llc Altered linolenic and linoleic acid content in plants
EP0771353A1 (de) * 1994-07-18 1997-05-07 Zeneca Limited Dna, konstruktionen, zellen und die davon abgeleiteten pflanzen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9806862A1 *

Also Published As

Publication number Publication date
BR9713462A (pt) 2000-03-28
CA2261577A1 (en) 1998-02-19
JP2001505409A (ja) 2001-04-24
CN1227609A (zh) 1999-09-01
AU4058497A (en) 1998-03-06
WO1998006862A1 (en) 1998-02-19

Similar Documents

Publication Publication Date Title
US6972351B2 (en) Methods for producing carotenoid compounds and specialty oils in plant seeds
WO1998006862A1 (en) Methods for producing carotenoid compounds and speciality oils in plant seeds
US6653530B1 (en) Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US20080276332A1 (en) Method for improving the agronomic and nutritional value of plants
EP1190068A2 (de) Nukleinsäuresequenzen für proteine, die an der tocopherolbiosynthese beteiligt sind
US8362324B2 (en) Nucleic acid sequences to proteins involved in tocopherol synthesis
AU2001253543B2 (en) Nucleic acid sequences to proteins involved in tocopherol synthesis
WO2001088169A2 (en) Method for producing carotenoid compounds in plants
AU2001253543A1 (en) Nucleic acid sequences to proteins involved in tocopherol synthesis
WO2001004330A1 (de) Identifizierung und überexpression einer dna-sequenz kodierend für eine 2-methyl-6-phytylhydrochinon-methyltransferase in pflanzen
EP1360309A2 (de) Nukleinsäuresequenzen für proteine, die an der tocopherolbiosynthese beteiligt sind
AU747542B2 (en) Methods for producing carotenoid compounds, and speciality oils in plant seeds
MXPA99001353A (en) Methods for producing carotenoid compounds and speciality oils in plant seeds
WO2004085656A2 (en) Enhanced accumulation of carotenoids in plants
WO2002103021A2 (de) Verfahren zur erhöhung des carotinoid-gehalts in transgenen pflanzen
DE19845231A1 (de) DNA-Sequenzen codierend für eine 1-Deoxy-D-Xylulose-5-Pphosphat Synthase, eine Hydroxyphenylpyruvat Dioxygenase und eine Geranylgeranylpyrophosphat Oxidoreduktase und deren Überproduktion in Pflanzen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990305

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20020517

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20021128