WO2001088169A2 - Procedes pour produire des composes de carotenoide, et huiles de specialite de graines de plantes - Google Patents

Procedes pour produire des composes de carotenoide, et huiles de specialite de graines de plantes Download PDF

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WO2001088169A2
WO2001088169A2 PCT/US2001/015264 US0115264W WO0188169A2 WO 2001088169 A2 WO2001088169 A2 WO 2001088169A2 US 0115264 W US0115264 W US 0115264W WO 0188169 A2 WO0188169 A2 WO 0188169A2
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carotenoid
gene
plant
phytoene
seeds
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WO2001088169A3 (fr
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Christine K. Shewmaker
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Monsanto Technology Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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

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 PPj to yield the first C 40 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 such 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 ingredients, as feed additives in animal 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. Consumption of ⁇ -carotene and lycopene has also been implicated as having preventative effects against certain kinds of cancers.
  • lutein consumption has been associated with prevention of macular degeneration of the eye.
  • 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,
  • 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, or a combination thereof.
  • 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.
  • 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, carotenoid mixtures.
  • transformed seed can provide a source for particular carotenoid compounds and/or for modified specialty oils having altered carotenoid compositions 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, SEQ ID No. 1.
  • Figure 2 presents constructs for expression of carotenoid biosynthesis genes in plant seeds.
  • Figure 2A shows plasmid pCGN3390 which con tains the napin promoter operably linked to the SSU/crtE sequence.
  • Figure 2B shows plasmid pCGN3392which contains the napin promoter operably linked to the SSU/crtE sequence.
  • Figure 2C shows plasmid pCGN9010 which contains the napin promoter operably linked to the SSU/crtl sequence.
  • Figure 2D shows plasmid pCGN9009 which contains the napin promoter operably linked to the SSU/crtE sequence and the napin promoter operably linked to the SSU/crtl sequence.
  • Figure 2 ⁇ shows plasmid pCGN9002 which contains the napin promoter operably linked to the SSU/crtE 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/crtE sequence and the napin promoter operably linked to an antisense beta cyclase sequence.
  • Figure 2G shows plasmid pCGN6204 which contains the napin promoter operably linked to the SSU/crtE sequence and the napin promoter operably linked to the SSU/crtW sequence.
  • Figure 2H shows plasmid pCGN6205 which contains the napin promoter operably linked to the SSU/crtE sequence and the napin promoter operably linked to the crtZ sequence.
  • Figure 21 shows plasmid pCGN6206 which contains the napin promoter operably linked to the SSU/crtE sequence, the napin promoter operably linked to the crtW sequence and the napin promoter operably linked to the crtZ sequence.
  • Figure 2J provides a schematic diagram of the corn expression construct pCGN9039.
  • 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 18:1 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 B. napus epsilon cyclase cDNA clone 9-4, SEQ LD No. 2.
  • FigurelO provides sequence of B. napus epsilon cyclase cDNA clone 7-6, SEQ ID No. 3.
  • Figure 11 provides sequence of a B. napus beta cyclase cDNA clone, SEQ ID No. 4.
  • Figure 12 provides T2 seed analysis of 3390 transformed Brassica napus plants.
  • Figure 13 provides T3 seed analysis of 3390 transformed Brassica napus plants.
  • Figure 14 provides T2 seed analysis of 9002 transformed Brassica napus plants.
  • Figure 15 shows the nucleotide sequence of the SSU/crtZ fusion sequence, SEQ LD No. 5, and the deduced amino acid sequence SEQ ID No. 6.
  • Figure 16 shows the nucleotide sequence of the SSU/crtW fusion sequence, SEQ ID No. 7, and the deduced amino acid sequence SEQ ID No. 8.
  • Figure 17 shows the HPLC trace for detection of xanthophylls from extractions from seed of 6204 transgenic lines.
  • Figure 18 provides the results of the expression of the maize phytoene synthase in Arabidopsis comparing the levels of B-carotene to total carotenoid levels in 9061 lines.
  • Figure 19 provides the complete nucleic acid sequence of the maize phytoene synthase sequence SEQ ID No. 9, and the deduced amino acid sequence, SEQ ID No. 10.
  • 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 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, or to further alter the fatty acid composition.
  • transformation 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. That is, there is an increase in the metabolic activity that can be further manipulated for the production of specific 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 significant increases in the levels 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 specialty 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 carotenoids. Furthermore, the carotenoid decomposition in the seeds of the present invention may be reduced significantly 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 (LPP) isomerase.
  • LPP isopentenyl diphosphate
  • a variety of sources are available for the early carotenoid biosynthesis genes and for the most part, a gene from any source can be utilized. However, it is recognized that because of co-suppression, the use of a plant gene native to the target host plant may not be desirable where increased expression of a particular enzyme is desired.
  • a number of early carotenoid biosynthesis genes also referred herein as DNA sequences derived from carotenoid biosynthesis gene coding regions, have been isolated and are available for use in the methods of the present invention. See, for example: IPP isomerase has been isolated from: R. Capsulatus (Hahn et al. (1996) J.
  • GenBank Accession Nos. U48963 and X82627 Clarkia xantiana GenBank Accession No. U48962, Arabidopsis thaliana GenBank Accession No. U48961, Schizosaccharmoyces pombe GenBank Accession No. U21154, human GenBank Accession No. X17025, Kluyveromyces lactis GenBank Accession No. X14230; geranylgeranyl pyrophosphate synthase from E. Uredovora Misawa et al. (1990)
  • 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.
  • 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, crtE ( 3392), or with phytoene desaturase, crtl (9010).
  • additional primary genes may be expressed to provide for even greater flux through the carotenoid pathway.
  • increased levels of phytoene are observed.
  • increasing the expression of phytoene desaturase as well as phytoene synthase may result in further increases in the levels of carotenoids, such as ⁇ - and ⁇ -carotene and lutein, produced.
  • carotenoids such as ⁇ - and ⁇ -carotene and lutein
  • Such plants may demonstrate even greater flux through the carotenoid pathway as indicated by the increased production of chlorophyll observed in plants of the present invention which have been transformed to express a GGPP synthase gene (crtE) in the absence of crtB overexpression.
  • crtE GGPP synthase gene
  • Plants expressing two or three different carotenoid biosynthsis genes may be produced by either transforming a plant with a construct providing for expression of the desired genes, using a multiple gene construct or by cotransformation with multiple constructs, or by crossing plants which contain the different desired genes.
  • 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.
  • 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 (cosuppression) or antisense orientation of the gene.
  • ⁇ -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 its derivative carotenoids, such as lutein.
  • overexpression of lycopene ⁇ -cyclase may be used to increase the accumulation of ⁇ -carotene derived carotenoids.
  • antisense lycopene epsilon cyclase and lycopene ⁇ -cyclase are examples of sequences which find use in secondary gene constructs of interest in the present invention.
  • increased expression of additional secondary genes may be desired for increased accumulation of a particular beta-carotene derived carotenoid.
  • increased ⁇ -carotene hydroxylase expression is useful for production of zeaxanthin
  • ⁇ -carotene hydroxylase and keto-introducing enzyme expression is useful for production of astaxanthin.
  • lycopene beta cyclase for accumulation of lycopene, 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.
  • the carotenoid pathway can be manipulated by expression of carotenoid biosynthesis genes to increase production of particular carotenoids, or by decreasing levels of a particular carotenoid by transformation with antisense DNA sequences which prevent the conversion of a selected precursor compound into the next carotenoid in the pathway.
  • ⁇ -carotene hydroxylase or crtZ (Hundle et al. (1993) FEBSLett. 315:329-334, GenBank Accession No. M87280) for the production of zeaxanthin; genes encoding keto-introducing enzymes, such as crtW (Misawa et al. (1995) J. Bacteriol. 177:6515-6584, WO 95/18220, WO 96/06172) or ⁇ -C-4-oxygenase (crtO; Harker and Hirschberg (1997) FEBSLett.
  • the pathway can be modified for the high production of any particular carotenoid compound of interest, or for a particular subset of carotenoid compounds, such as xanthophylls.
  • carotenoid compounds such as xanthophylls.
  • Such compounds include but are not limited to the particular compounds described above, as well as, ⁇ -cryptoxanthin, ⁇ -cryptoxanthin, ⁇ - carotene, phytofluene, neurosporane, adonixanthin, echmeneone, hydroxycanthaxanthm and the like.
  • any compound of interest in the carotenoid pathway can be produced at high levels in a seed.
  • Secondary genes can also be selected to alter the fatty acid content of the plant for the production of specialty 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 mangosteen acyl-ACP thioesterase as a secondary gene for fatty acid content modification.
  • a high stearate content may be obtained in seeds by expression of a mangosteen acyl-ACP thioesterase.
  • 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 either by inclusion of both expression constructs in a single transformation vector or by using separate vector, each of which express desired primary or secondary genes.
  • 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.
  • tissue specific promoters the carotenoid levels can be altered in particular tissues of the plant.
  • carotenoid levels in the seed 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 imtiation 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 imtiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination 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 genes of interest of the present invention will be targeted to plastids, such as chloroplasts, for expression.
  • the carotenoid biosynthesis gene or genes of interest may be inserted into the plastid for expression with appropriate plastid constructs and regulatory elements.
  • nuclear transformation may be used in which case the expression cassette will contain a gene encoding a transit peptide to direct the carotenoid biosynthesis 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) J Biol. Chem. 264:11544-11550; della-Cioppa et al.
  • Plant carotenoid genes useful in the invention may utilize native or heterologous transit peptides.
  • the gene or DNA sequence of interest is an antisense DNA
  • targeting to a plastid is not required.
  • antisense inhibition of a given carotenoid biosynthesis gene is desired, the entire DNA sequence derived from the carotenoid biosynthesis gene is not required.
  • the construct may also include any other necessary regulators such as plant translational consensus sequences (Joshi, C.P., (1987), 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.
  • 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
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G., and Sarnow, P., (1991), Nature, 353:90-94; untranslated leader from the coat protein rriRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L., (1987), Nature, 325:622-625; tobacco mosaic virus leader (TMV), (Gallie, D.R. et al.,
  • the sequence of interest may be expressed 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 55:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17: 477-498. In this manner, the nucleotide sequences can be optimized for expression in any plant.
  • in vitro mutagenesis primer repair, restriction, annealing, resection, ligation, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and transversions, may be involved.
  • the recombinant 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 5 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 53:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology 5:915-921) and ballistic particle
  • a plant plastid can be transformed directly. Stable transformation
  • 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. In such methods, plastid
  • 25 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 silent 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.
  • Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci., USA 97:7301-7305.
  • 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 (1986), 5:81-84. These plants may then be grown, and either self or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may. be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. As a host cell, any plant variety may be employed. Of particular interest, are plant species which provide seeds of interest.
  • seed 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. wheat, barley, oats, amaranth, flax, rye, triticale, rice, corn, etc.; other edible seeds or seeds with edible parts including pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, radish, alfalfa, cocoa, coffee, tree nuts such as walnuts, almonds, pecans, chick-peas etc.
  • oil seeds such as oilseed Brassica seeds, cotton seeds, soybean, safflower, sunflower, coconut, palm, and the like
  • grain seeds e.g. wheat, barley, oats, amaranth, flax, rye, triticale, rice, corn, etc.
  • other edible seeds or seeds with edible parts including pumpkin, squash, sesame, poppy, grape, mung beans, peanut,
  • ⁇ -carotene levels being 20 fold higher than ⁇ -carotene levels.
  • a second early carotenoid biosynthesis gene such as phytoene desaturase, may be used with crtB to increase the metabolic flux through the carotenoid/ isoprenoid pathway in cotton to produce a particular carotenoid.
  • the methods of the present invention have also been demonstrated herein to provide increased carotenoid production in additional plant species, such as Arabidopsis and corn.
  • a second early carotenoid biosynthesis gene may be used with crtB to increase the metabolic flux through the carotenoid/ isoprenoid pathway in cotton to produce a particular carotenoid and to reduce the increased levels of phytoene.
  • a second early carotenoid biosynthesis gene can be employed with the crtB to increase the metabolic flux through the carotenoid/ isoprenoid pathway in corn to produce a particular carotenoid and to reduce the increased levels of phytoene.
  • additional genes, including secondary carotenoid biosynthesis genes can also be used to direct the production of particular carotenoids and xanthophyls.
  • seed transcriptional imtiation 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.
  • genes can be selected to promote the formation of compounds of interest.
  • the gene selected is an early carotenoid biosynthesis gene
  • the transformed seed has a significant increase in carotenoid biosynthesis as the result of an increase in the flux through the pathway.
  • For 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. Seeds obtained from corn plants transformed with an early carotenoid biosynthesis gene also demonstrate an increased amount of carotenoid production.
  • 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, including phytoene.
  • ⁇ - 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.
  • GGPP synthase or phytoene desaturase 1.5 to 2 fold increases in lutein and ⁇ -carotene have been obtained in at least one transgenic plant for each gene. Lycopene is also detected in seeds from Brassica napus plants transformed with a crtE (GGPP synthase) gene. Total carotenoids in crtE or crtl transformants are also increased approximately 2 fold. Chlorophyll levels are also increased in B. napus transgenic plants expressing a crtE gene suggesting an increase in the levels of geranylgeranyl pyrophosphate (GGPP), which is the branch point substrate for carotenoid, chlorophyll and tocopherol biosynthesis.
  • GGPP geranylgeranyl pyrophosphate
  • phytoene synthase is cofransformed into Brassica napus with a second early carotenoid biosynthesis gene, phytoene desaturase
  • significant increases of particular carotenoids include increases in ⁇ -carotene, ⁇ -carotene, and lutein such as observed by expression of crtB alone.
  • lycopene and phytoene levels are also increased in such plants, but increases are difficult to quantitate as these levels are too low to be detected in untransformed Brassica napus plants.
  • total carotenoid levels greater than those observed with crtB alone may be obtained.
  • total carotenoid levels of 1.5 fold those observed in crtB plants were obtained.
  • Lycopene levels are also increased over levels obtained in seeds of plants transformed with crtB alone. Lycopene levels may be increased from 4 to 15 fold over those obtained in seed of a homozygous crtB plant.
  • a reduction in the ratio of phytoene to total carotenoids is also obtained, and as a result, levels of ⁇ -carotene and ⁇ -carotene are increased 1.2 to 1.8 fold over those obtained with crtB alone.
  • phytoene levels constituted as much as 20%» of total carotenoids, while in plants cotransformed with phytoene synthase and phytoene desaturase, phytoene levels represent only 4% to 7% of the total carotenoids.
  • 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. Therefore, significant amounts of carotenoids of interest can be produced in the transformed seeds of the present invention.
  • phytoene synthase is cofransformed with a secondary carotenoid biosynthesis gene, ⁇ -carotene ketolase
  • increases in levels of ⁇ -carotene, ⁇ -carotene and phytoene are obtained.
  • echinenone and canthaxanthin levels are also increased.
  • increases are difficult to quantitate as echinenone and canthaxanthin are either not produced in Brassica napus , or the levels are too low to be detected in B. napus plants expressing phytoene synthase alone and nontransformed control plants.
  • a third carotenoid biosynthesis gene such as ⁇ -carotene hydroxylase (crtZ)
  • a fourth carotenoid biosynthesis gene such as phytoene desaturase, may also find use in the present invention.
  • the carotenoid echinenone is a reaction intermediate in the production of canthoxanthin from ⁇ -carotene.
  • the ⁇ -carotene ketolase (crtW) could react with the ⁇ -ring of ⁇ - or ⁇ -carotene.
  • One ⁇ -ring reaction in ⁇ -carotene results in echinenone, two ⁇ -ring reactions in ⁇ -carotene form canthaxanthin, and one ⁇ -ring reaction in ⁇ -carotene makes 4-keto- ⁇ -carotene.
  • This enzyme can not react with the ⁇ - ring of ⁇ -carotene.
  • two additional peaks on the HPLC chromatogram are produced in similar amounts, one representing echinenenone, and the other may represent 4-keto- ⁇ -carotene.
  • phytoene synthase is cofransformed with an antisense secondary carotenoid biosynthsis gene, ⁇ -cyclase, large increases in levels of ⁇ -carotene, ⁇ -carotene and phytoene, such as those seen with transformation with crtB alone, are obtained. Some difference in the ratio of ⁇ -carotene to ⁇ -carotene is observed as compared to plants transformed with crtB alone, but large increases in both ⁇ -carotene and ⁇ -carotene levels are still observed. Lutein levels, on the other hand, are either unchanged, increased, or in some cases decreased by as much as 80% as compared to seeds of untransformed control plants.
  • Initiation of carotenoid biosynthesis begins at approximately 15 days post anthesis in B. napus seeds, while expression of transformed genes utilizing the napin 5 promoter begins about 18 days post anthesis.
  • an earlier promoter such as that of the Lesquerella kappa hyrodoxylase ( described in pending U.S. patent application 08/898,038, filed 18 July, 1997), may find use.
  • an earlier promoter such as that of the Lesquerella kappa hyrodoxylase ( described in pending U.S. patent application 08/898,038, filed 18 July, 1997), may find use.
  • an earlier promoter such as that of the Lesquerella kappa hyrodoxylase ( described in pending U.S. patent application 08/898,038, filed 18 July, 1997), may find use.
  • the Lesquerella kappa hyrodoxylase described in pending U.S. patent application 08/898,038, filed 18 July, 1997)
  • L 0 antisense an earlier seed specific tanscriptional initiation region, may be used with a secondary carotenoid biosynthesis gene.
  • the seeds of the invention which have been transformed with the primary early carotenoid biosynthesis gene also provide a source for novel oil compositions.
  • L5 oleic acid content in seed oil By 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,
  • 20 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 75%.
  • 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 high ⁇ - and ⁇ -carotene oils would have a longer shelf life as both the oleic acid and ⁇ - and ⁇ -carotene 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 transformed seed and embryos additionally find use as screenable markers.
  • 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 examples.
  • EXPERIMENTAL Example 1 Expression Construct and Plant Transformation
  • 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 BgUl 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/crtE 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.
  • the SSU leader and crtZ gene sequences were joined by PCR .
  • the crtZ gene 5 (Misawa, et al. (1995) supra) nucleotide sequence was resynthesized to adjust for plant codon usage.
  • the re synthesized crtZ gene was joined to the SSU leader by PCR as follows. A Bglll site was included upstream of the SSU leader translation start site and a Xhol site was included downstream of the crtZ stop codon to facilitate cloning in the napin expression cassette.
  • the nucleotide sequence of the complete ssu rtZ fusion is L 0 shown in Figure 15.
  • the SSU leader and crtW gene sequences were joined by PCR .
  • the crtW gene (Misawa, et al. (1995) supra) nucleotide sequence was resynthesized to adjust for plant codon usage.
  • the re synthesized crtW gene was joined to the SSU leader by PCR as L 5 follows.
  • a Bglll site was included upstream of the SSU leader translation start site and a Xhol site was included downstream of the stop codon to facilitate cloning in the napin expression cassette.
  • the nucleotide sequence of the complete ssu:crtW fusion is shown in Figure 16.
  • pCGN1559PASS is a binary vector for -4grob ⁇ ctertwm-mediated transformation such as those described by McBride et al. (Plant Mol. Biol. (1990) 14:269-216) and is prepared from pCGN1559 by substitution of 5 the pCGNl 559 linker region with a linker region containing the following restriction digestion sites: AsplWAscllPacVXball BammiSwaVSse8381(PstT)IHindRl.
  • a map of pCGN3390 is provided in Figure 2A.
  • the crtB coding sequence from E. herbicola (Application WO 91/13078, Armstrong et al. (1990) supra) was cloned to be expressed under control from
  • This cassette also includes the transcriptional termination region downstream of the cloning site of nopaline synthase, nos 3' (Depicker et al., J. Molec. Appl. Genet. (1982) 1 : 562-573) to create the vector pCGN9039 ( Figure 2J) for transformation into corn.
  • a map of pCGN9010 is provided in Figure 2C.
  • Brassica napus epsilon cyclase genes are isolated by PCR using primers L0 designed from an Arabidopsis epsilon cyclase gene (Cunningham FX Jr (1996) Plant
  • B. napus epsilon cyclase genes Sequence of B. napus epsilon cyclase genes is provided in Figures 9 (clone 9-4) and 10 (clone 7-6).
  • An antisense construct is prepared by cloning snXh ⁇ UBamHl fragment of cDNA clone 9-4 into a napin expression cassette (pCGN3223) digested withNf ⁇ oI and Bglll.
  • the napin 5'-antisense epsilon cyclase- L5 napin 3' fragment is cloned along with a napin 5'-SSU/crt7?-napin 3' fragment, fragment into a binary vector for plant transformation, resulting in pCGN9002, shown in Figure 2E.
  • Brassica napus beta cyclase genes are isolated by PCR using primers designed 20 from an Arabidopsis beta cyclase gene (Cunningham FX Jr (1996) Plant Cell 5:1613-
  • Sequence of a B. napus beta cyclase cDNA, 32-3, is provided in Figures 11.
  • An antisense construct is prepared by cloning XhoI fragment of the beta cyclase cDNA clone into a napin expression cassette (pCGN3223) digested withJ ⁇ oI.
  • a clone containing the beta cyclase in the antisense orientation is selected.
  • the napin 5'- 25 antisense beta cyclase-napin 3' fragment is cloned along with a napin 5'-SSU/crt7?-napin 3' fragment into a binary vector for plant transformation, resulting in pCGN9017, shown in Figure 2F.
  • the vector pCGN9003 was constructed by removing the restriction sites between the crtB coding sequence and the napin 3' sequence by digestion with Clal and Xhol and filling the ends with klenow creating the vector pCGN9000.
  • PCGN9000 was digested with-4sp718, and the fragment containing the napin 57SSU:crtB/napin 3' was ligated into the binary vector pCGN5139.
  • 35S transcriptional initiation region (35S 5') and transcription termination (35S 3') sequences (Fraley et al., Proc. N ⁇ tl. Ac ⁇ d. Sci (1983) 80:4803-4807, Gardner et ⁇ l, (1986) Plant Mol Biol 6:221-228).
  • the 35S 5'-nptII-35S 3' fragment was then cloned into a vector containing ori322, Right border (0.5Kb), lacZ, Left Border (0.58Kb), as an Xlio I fragment between the Right border-lacZ and Left border sequences.
  • the plastid targeted ssu rtZ fusion was cloned into the napin pCGN3223 seed expression cassette as a Bgl II -Xho I fragment to generate pCGN6203.
  • the plasmid pCGN6203 carrying the complete napin cassette with ssu:crtZ was digested with Notl to excise the napin cassette containing the ssu:crtZ coding region.
  • the excised fragment was ligated into the Not I site of the binary pCGN9003 carrying the napin SSU rtB construct.
  • the resulting construct, pCGN6205 ( Figure 2H) is a binary vector for Agrobacterium-mediated transformation such as those described by McBride et al.
  • the ssu crtW plastid targeted fusion was cloned into the napin pCGN3223 seed expression cassette as a Bgl II -Xho I fragment to generate plasmid pCGN6202.
  • the plasmid pCGN6202 carrying the napin cassette with ssu rtW was digested with Notl to excise a DNA fragment containing the napin cassette with ssu:crtZ.
  • the resulting fragment was ligated into the Not I site of the binary pCGN9003 (described above) carrying the SSU:crtB napin construct .
  • the resulting pCGN6204 ( Figure 2G) is a binary vector for Agrobacterium-mediated transformation such as those described by McBride et al. (Plant Mol. Biol. (1990) 14:269-276) and is prepared from pCGN1559 by substitution of the pCGN1559 linker region with a linker region containing the following restriction digestion sites: Asp718/Ascl/Pacl/Xbal/
  • Transformed cotton plants, Gossypium hirsutum, containing phytoene synthase may be obtained using methods described in issued U.S. patent No. 5,004,863, and
  • Transgenic Arabidopsis thaliana plants containing phytoene synthase may be obtained by Agrobacterium-mediated transformation as described by Valverkens et ⁇ l, (Proc. Nat. Acad. Sci. (1988) 55:5536-5540), or as described by Bent et al. ((1994),
  • Microprojectile bombardment methods such as described by Klein et al. (Bio/Technology 70:286-291) may also be used to obtain nuclear transformed plants.
  • 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. At this point in the extraction, the 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 AIM 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 ATM extracts were then washed two more time with 400 ⁇ l of ether, and the ether fractions from the three ATM 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 lOO ⁇ 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.
  • 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 initial solution was 70:20:10 (A:B:C). At 2.5 minutes the solution is ramped over 5 minutes to 65:25:10 (A:B:C) and held at this for 12.5 minutes.
  • the solution is then ramped to 70:20:10 (A:B:C) over two minutes followed by a three minute delay prior to injection of the next sample.
  • the absorbance of the eluting samples is continuously monitored at 450 and 280 nm and known chemical and biological standards were used to identify the various absorbance peaks.
  • Mature 3390 T2 seed were sent to an analytical laboratory for quantitative analysis using standard HPLC methods known in the art. These results of these analysis are shown in Table 4 below. Compound levels are presented as ⁇ g/g. Seeds designated "Maroon” were selected based on seed color. The seeds which have orange embryos appear maroon colored at maturity as opposed to the black-brown appearance of seeds from wild type plants of this cultivar. Seeds designated as "Random” were not selected for color. As 3390-1 is segregating 3 to 1 for Kan, the "Random" population includes a proportion of nulls. The maroon population contains only fransgenics. Due to an effort to exclude nulls from this population, the inclusion of homozygotes may be favored. TABLE 4
  • transgenic sample in the non-transgenic sample, “other” includes mostly very polar compounds, such as neoxanthin, violaxanthin, etc. In the transgenic sample “ other” includes these and additional compounds, such as zeta-carotene, neurosporene, and mono-cyclic carotenoids.
  • 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 (SPOOl-1). The results of these analyses are provided in Table 5 below as weight %> total fatty acids.
  • Carotenoids were analyzed in mature T2 seeds of 3392 B. napus plants franformed to express the E. uredovora crtE gene. Approximately two fold increases 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 untransformed control plants. Analysis of seeds from 7 additional 3392 transformants did not reveal significant increases in the carotenoid levels. G. Analysis of Chlorophyll and Tocopherol Levels in crtE Transgenic Plants
  • Chlorophyll levels were analyzed using a spectrophotometric assay (Bruinsma, J. 1961, A comment on the spectrophotometric determination of chlororphyll, Biochem Biophy Acta, 52:576-578) in mature T2 seeds of transgenic 3392 B. napus plants.
  • Chlorophyll concentrations of the 35 DPA seeds of two lines were increased by approximately 60% compared to the levels of the control plant.
  • the initial results 30 demonstrate that the GGPP synthase gene increased the GGPP substrate availability for chlorophyll biosynthesis during seed development. Mature seeds of the 3392-16 line had higher chlorophyll and carotenoid concentrations than those of the control.
  • Carotenoid levels of Mature 9009 T2 seeds were extracted and quantified on an HPLC as follows. Approximately lOOmg of seeds were ground in a mortar and pestle in 5 3ml extraction solvent ( hexane/acetone/ethanol ( 50/25/25 v/v) with 0.2ml of an internal standard ( 5mg/ml ⁇ -apo-8 " carotenal ( dissolved in lOO ⁇ l hexane), in acetonitrile/methylene chloride/methanol ( 50/40/10, v/v/)). The extraction solution was transferred to a new glass tube, and the remaining seed was again extracted with the extraction solvent and pooled with first extraction solution. The extraction was repeated
  • phytoene desaturase can have a synergistic effect with phytoene synthase in increasing the metabolic flux through the carotenoid/ isoprenoid pathway, and provides for even greater increases in a desired carotenoid compound, such as ⁇ -carotene and ⁇ -carotene, than is obtained by expression of crtB alone.
  • the increased flux also appears to result in increased total carotenoid production, in addition to the composition shift from phytoene.
  • the carotenoid levels in the segregating T2 seed populations of 9009-10 are significantly higher than those detected in the 3390 homozygous seed population in 3390-1-6-15.
  • An approximately 3 fold increase in lutein was observed in seeds of plant 3390- C130-5-1.
  • Alpha-carotene, ⁇ -carotene and phytoene were also observed in this line and are undetectable in nonfransformed control plants. With ⁇ -carotenoid levels being 20 fold higher than those of ⁇ -carotene. Total carotenoid levels were increased by more than 250 fold, with phytoene accounting for approximately 80% of that total.
  • Carotenoid levels of Mature 6204 T2 seeds were extracted and quantified on an HPLC as follows. Approximately lOOmg of seeds were ground in a mortar and pestle in 5 3ml extraction solvent ( hexane/acetone/ethanol ( 50/25/25 v/v) with 0.3ml of an internal standard ( 5mg/ml ⁇ -apo-8' carotenal ( dissolved in lOO ⁇ l hexane), in acetonitrile/methylene chloride/methanol ( 50/40/10, v/v/)). The extraction solution was transferred to a new glass tube, and the remaining seed was again extracted with the 2 ml extraction solvent and pooled with first extraction solution. The extraction was repeated
  • results demonstrate that as with plants transformed to express crtB alone, plants expressing crtB and crtW contain significant increases in total carotenoid levels. Furthermore, the results show an increase in the levels of canthaxanthin, when compared to the levels obtained from seeds of plants transformed with crtB alone, as well as nonfransformed control plants. In addition, other products were also produced in plants expressing crtB and crtW. Increased levels of echineone, a reaction intermediate, as well as a putative 4-keto- ⁇ -carotene (Figure 17).
  • Transgenic Arabidopsis expressing the maize phytoene synthase demonstrate an increase in total carotenoid levels.
  • transgenic Arabidopsis lines containing the maize phytoene synthase produce less phytoene as a percentage of total carotenoids.
  • the 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.

Abstract

Cette invention se rapporte à des procédés servant à produire des plantes et des graines ayant des compositions de caroténoïde modifiées, en transformant les plantes hôtes avec des produits de synthèse ayant une région d'initialisation transcriptionnelle à partir d'un gène exprimé dans une graine de plante, un peptide de transition de plastide, une séquence d'ADN dérivée d'au moins une région de codage de gène de biosynthèse de caroténoïde, et une région de terminaison transcriptionnelle. Ces procédés sont particulièrement utiles pour augmenter la teneur en caroténoïde de plantes à graines oléagineuses.
PCT/US2001/015264 2000-05-12 2001-05-11 Procedes pour produire des composes de carotenoide, et huiles de specialite de graines de plantes WO2001088169A2 (fr)

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US7605244B2 (en) 2001-08-17 2009-10-20 Monsanto Technology Llc Gamma tocopherol methyltransferase coding sequence from Brassica and uses thereof
WO2004018688A1 (fr) * 2002-08-20 2004-03-04 Sungene Gmbh & Co. Kgaa Procede de preparation de $g(b)-carotinoides
US7663021B2 (en) 2002-12-06 2010-02-16 Del Monte Fresh Produce Company Transgenic pineapple plants with modified carotenoid levels and methods of their production
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
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EP3792353A1 (fr) * 2019-09-13 2021-03-17 CRAG - Centre de Recerca en Agrigenomica CSIC-IRTA-UB-UAB Biogenèse chromoplaste artificielle
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