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Definitions

• the present invention relates to a process for the preparation of zeaxanthin and / or its biosynthetic intermediates and / or secondary products by cultivating genetically modified plants which, compared to the wild type, have a reduced ⁇ -cyclase sequence caused by double-stranded ⁇ -cyclase-ribonucleic acid sequences. Have activity, the genetically modified plants, and their use as food and feed and for the production of carotenoid extracts. , "
• the ⁇ -cyclase activity in plants can be reduced compared to the wild type, for example by reducing the amount of ⁇ -cyclase protein or the amount of ⁇ -cyclase mRNA in the plant. Accordingly, ⁇ -cyclase activity which is reduced compared to the wild type can be determined directly or by determining the amount of ⁇ -cyclase protein or the ⁇ -cyclase mRNA merige of the plant according to the invention in comparison to the wild type.
• ⁇ -cyclase activity in genetically modified plants according to the invention and in wild-type or reference plants is preferably carried out under the following conditions:
• plant can be understood to mean the starting plant (wild type) or a genetically modified plant according to the invention or both.
• the ⁇ -cyclase activity is reduced by introducing at least one double-stranded ⁇ -cyclase ribonucleic acid sequence, hereinafter also called ⁇ -cyclase dsRNA, or an expression cassette or expression cassette ensuring its expression in plants.
• ⁇ -cyclase dsRNA double-stranded ⁇ -cyclase ribonucleic acid sequence
• the ratio of double-stranded molecules to corresponding dissociated forms is preferably at least 1 to 10, preferably 1: 1, particularly preferably 5: 1, most preferably 10: 1.
• b) is identical to at least part of the plant's own ⁇ -cyclase promoter sequence.
• RNA the "at least a part of the plant's ⁇ -cyclase promoter sequence identical" is preferably meant that the RNA sequence with at least a portion of the theo- retical transcript 15 'of the ⁇ -cyclase promoter Sequence, .also the corresponding RNA sequence, is identical.
• the ⁇ -cyclase dsRNA therefore contains a sequence which is identical to a part of the plant's own ⁇ -cyclase transcripts and which codes for the 5th ⁇ end or the 3 ⁇ end of the plant's own nucleic acid contains an ⁇ -cyclase.
• non-translated areas are in the 5 * or
• the ⁇ -cyclase-nucleic acid sequence or the corresponding transcript is preferred
• an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of an ⁇ -cyclase gene transcript, for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50 ° C or 70 ° C for 12 to 16 h).
• the present invention therefore also relates to a “ nucleic acid construct that can be transcribed into
• RNA strand comprising at least one ribonucleotide sequence which is essentially identical to at least part of the “sense” RNA ⁇ -cyclase transcript
• RNA strand which is essentially — preferably completely — complementary to the RNA sense strand under a).
• the following partial sequences are particularly preferably used, in particular for Tagetes erecta:
• SEQ. ID. NO. 6 Sense fragment of the 5'-terminal region of the ⁇ -cyclase
• SEQ. ID. No. 14 Antisense fragment of the ⁇ -cyclase promoter
• the dsRNA can be introduced in an amount that enables at least one copy per cell. Larger quantities (e.g. at least 5, 10, 100, 500 or 1000 copies per cell) can possibly result in an efficient reduction.
• a plant selected from the plant genera Marigold, Tagetes, Acacia, Aconituim, Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia, Calendula, Caltha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum, Citrus is particularly preferably used as the plant , Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus, Dimorphotheca, Doronicum,
• the transgenic plants are grown on nutrient media in a manner known per se and harvested accordingly.
• Zeaxanthin and / or its biosynthetic intermediate and / or secondary products are isolated from the harvested petals in a manner known per se, for example by drying and subsequent extraction and, if appropriate, further rer chemical or physical "" cleaning processes, such as precipitation methods, crystallography, thermal separation processes, such as rectification processes or physical separation processes, such as chromatography.
• Zeaxanthin and / or its biosynthetic intermediate and / or secondary products are isolated from the petals, for example, preferably by organic solvents such as acetone, hexane, ether or tert. Methyl butyl ether.
• ketocarotenoids in particular from petals, are described, for example, in Egger and Kleinig (Phytochemistry (1967) 6, 437-440) and Egger (Phytochemistry (1965) 4, 609-618).
• Pathogen-inducible promoters include those of genes induced by pathogen attack such as genes from PR proteins, SAR proteins, b-1, 3-glucanase, chitinase etc. (e.g. Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant Cell 4: 645-656; Van Loön (1985) Plant Mol Viral 4: 111-116; Marineau et.al. (1987) Plant Mol -Biol 9: 335-342; Matton et al ' ._ (1987) Molecülar Plant-Microbe Interactions 2: 325-342; Somssich et al.
• wound-inducible engines such as the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 genes (US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin (McGurl et al. (1992) Science 225: 1570-1573), the WIPl gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Ekelkamp et al. (1993) FEBS Letters 323: 73-76), the MPI gene (Corderok et al. (1994) The Plant J 6 ( 2) .141-150) and the like.
• the pinll gene Rost al. (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494
• promoters are particularly preferred which ensure expression in tissues or parts of plants in which, for example, the biosynthesis of ketocarotenoids or their precursors takes place.
• promoters with specificities for the anthers, ovaries, petals, sepals, flowers, leaves, stems and roots and combinations thereof are preferred.
• Tuber-, storage root- or root-specific promoters are, for example, the " Class I Patatin Promoter (B33) or the potato cathepsin D inhibitor promoter.
• the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. " Acids Res. 12 (1984), 8711) " or particularly preferably pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).
• transgenic plants can be regenerated in a known manner which contain a gene encoding a ketolase for the expression of a nucleic acid encoding the expression cassette.
• an expression cassette is incorporated as an insert into a recombinant vector whose vector DNA comprises additional functional regulatory signals, examples play example sequences for replication or integration. Suitable vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993).
• the genetically modified plants can, as described above, for the production of zeaxanthin and / or its biosynthetic intermediates and / or secondary products, in particular for the production of lycopene, ⁇ -carotene, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3 'Hydroxyechinenone, adonirubin or adonixanthin and in particular for the production of astaxanthin can be used.
• an increased content is also understood to mean a caused content of ketocarotenoids or astaxanthin.
• Genetically modified plants according to the invention which are edible by humans and animals and have an increased content of zeaxanthin and / or its biosynthetic intermediate and / or secondary products can also be used, for example, directly or after processing known per se as food or feed or as feed and food supplements , Furthermore, the genetically modified plants can be used for the production of carotenoid-containing extracts of the plants and / or for the production of feed and food supplements.
• the genetically modified plants can ⁇ . can also be used as ornamental plants in the horticulture sector.
• FIG. 5 is not restricted to these:
• inverted repeat transcripts consisting of - . Fragments of the epsilon cyclase in Tagetes erecta were carried out under the control of a modified version AP3P of the flower-specific promoter AP3 from Arabidopsis thaliana (AL132971: nucleotide region 25 9298-10200; Hill et al. (1998) Development 125: 1711-1721)
• the PCR for the amplification of the DNA encoding the AP3 promoter fragment was carried out in a 50 1 reaction mixture which contained: 45 - 1 ul genomic DNA from ' A. tanne.liana (1: 100 dil prepared as described above) 0.25 mM dNTPs
• the 922 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (invi rogen) using standard methods
• Sequencing of the clone pTAP3 confirmed a sequence consisting only of an insertion (a G in position 9765 of the sequence AL132971) and a base exchange (a G instead of an A in position 9726 of the sequence AL132971) from the published AP3 sequence (AL132971 , Nucleotide region
• the modified version AP3P was produced by means of recombinant PCR using the plasmid pTAP3.
• the region 10200-9771 was amplified with the primers PR7 (SEQ ID No. 15) and primers PR9 (SEQ ID No. 17) (amplificate A7 / 9), the 35 region 9526-9285 with the PR8 (SEQ ID No. 16). and PR10 (SEQ ID No. 18) amplified (amplificate A8 / 10).
• the cloning was carried out by isolating the 771 bp SacI-HindIII fragment from pTAP3P and ligating into the SacI-HindIII cut vector pJITH7.
• the clone that contains the AP3P promoter instead of the original d35S promoter is called pJAP3P.
• the PCR conditions were as follows:
• the PCR for the amplification of the sequence of the intron PIV2 of the ST-LS1 gene was carried out in a 50 1 reaction which contained: - 1 ⁇ l p35SGUS INT
• PCR amplification using primers PR42 and PR43 resulted 'in a 443 bp fragment
• PCR amplification using primers PR44 and PR45 resulted in a 444 bp fragment.
• a CHRC promoter fragment using genomic DNA from petunia (prepared according to standard methods) and the primers PRCHRC5 (SEQ ID No. 42) and PRCHRC3 (SEQ ID No. 43) amplified.
• the amplificate was cloned into the cloning vector pCR2.1 (Invitrogen). Sequencing of the resulting clone pCR2.1-CHRC with the primers M13 and T7 confirmed a sequence identical to the sequence AF099501. This clone was therefore used for the cloning into the expression vector pJAl3.
• Total RNA was prepared from Tagetes flowers as described in Example 2.
• the PCR for the amplification of the PR46-PR457 DNA fragment which contains the 3'-terminal 384 bp region of the epsilon cyclase, was carried out in a 50 ⁇ l reaction mixture which contained:
• PCR amplification with SEQ ID No.26 and SEQ ID No. ' 27 resulted in a 392 bp fragment
• the PCR amplification with SEQ ID No.28 and SEQ ID No. 29 resulted in a 396 bp fragment.
• the first cloning step was carried out by isolating the 396 bp PR48-PR49 BamHI-EcoRI fragment from the cloning vector pCR-BluntII (Invitrogen) and ligation with the BamHI-EcoRI cut vector pJAIl.
• the clone that contains the terminal region of the epsilon cyclase in the antisense orientation is called pJAl4.
• the ligation results in a transcriptional fusion between the antisense fragment of the 3'-terminal region of the epsilon cyclase and the polyadenylation signal from CaMV.
• fragment AP3P contains the modified AP3P promoter (771 bp), fragment sense the 3 'region of the Epsilon cyclase from Tagetes erecta . (435 bp) in sense orientation, fragment intron the intron ' IV2 of the potato gene ST-LSI / fragment anti the 3' region of the Epsilon cyclase from Tagetes erecta (435 bp) in "antisense orientation, and fragment term ( 761 bp) the poly-adenylation signal of CaMV.
• the PCR for the amplification of the PR50-PR51 DNA fragment which contains, among other things, the 312 bp promoter fragment of epsilon cyclase, was carried out in a 50 ⁇ l reaction mixture which contained:
• the amplificate was cloned into the PCR cloning vector pCR2.1 (Invitrogen) using standard methods. Sequencing with the primers M13 and T7 resulted in the sequence SEQ ID No. 11. This sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Tagetes erecta Orange Prince line used.
• ADl initially represented a mixture of primers of the sequences (a / c / g / t) tcga (g / c) t (a / t) t (g / c) g (a / t) gtt.
• the PCR reaction TAIL1 was carried out under the following cycle conditions:
• the TAIL3-PCR was carried out in a 100 ⁇ l reaction mixture, which contained:
• the PCR reaction TAIL3 was carried out under the following cycle conditions; 20X 94 ° C: 15 seconds, 29 ° C: 30 seconds, 72 ° C: 2 minutes IX 72 ° C: 5 minutes
• the amplificate was cloned into the PCR cloning vector pCR2.1 (Invitrogen) using standard methods. Sequencing with the primers Ml3 and T7 resulted in the sequence SEQ ID No. 12. This sequence is identical to the ecyclase region within the sequence SEQ ID No. 11, which was isolated with the IPCR strategy, and thus represents the nucleotide sequence in the Tagetes erecta line Orange Prince used.
• the promoter fragments were PCR by means of plasmid DNA (clone pTA-ecycP, see Example 4) and the primers PR124 (SEQ ID No. 36) and PR126 (SEQ ID No. 38) or the primer PR125 (SEQ ID No 37) and PR127 (SEQ ID No. 39).
• the conditions of the PCR reactions were as follows: The PCR for the amplification of the PR1 4-PR12.6_ DNA fragment, which contains the promoter fragment from the epsilon cyclase, was carried out in a 50 ⁇ l reaction mixture which contained:
• PCR amplification with primers PR124 and PR126 resulted in a 358 bp fragment
• PCR amplification with primers PR125 and PR127 resulted in a 361 bp fragment.
• the two amplicons were cloned into the PCR cloning vector pCR-BluntII (Invitrogen) using standard methods. Sequencing with the primer SP6 each confirmed a sequence which, apart from the restriction sites introduced, is identical to SEQ ID No. 11. These clones were therefore used for the production of an inverted repeat construct in the cloning vector pJAII (see example 1).
• the first cloning step was carried out by isolating the 358 bp PR124-PR126 HindIII-SalI fragment from the cloning vector pCR-BluntII (Invitrogen) and ligation with the BamHI-EcoRI cut vector pJAII.
• the clone that contains the epsilon cyclase promoter fragment in the sense orientation is called cs43.
• the sense fragment of the epsilon cyclase promoter is inserted between the AP3P promoter and the intron by the ligation.
• the second cloning step was carried out by isolating the 36 lbp PR125-PR127 BamHI-EcoRI fragment from the cloning vector pCR-BluntII (Invitrogen) and ligation with BamHI-EcoRI cut vector cs43.
• the clone that contains the epsilon cyclase promoter fragment in the antisense orientation is called cs44.
• the ligation creates a transcriptional fusion between the intron and antisense fragments of the epsilon cyclase promoter.
• the AP3P promoter was cloned in cs45 in antisense orientation at the 3'terminus of the epsilon-cyclase antisense fragment.
• the AP3P promoter fragment from pJAII was amplified using the primers PR128 and PR129.
• the amplificate was cloned into the cloning vector pCR2.1 (Invitrogen).
• the sequencing with the primers M13 and T7 confirmed a sequence SEQ ID No. 1 identical sequence ..
• the expression vectors for the Agrobacterium -mediated transformation of the AP3P-controlled inverted repeat transcript in Tagetes erecta were produced using the binary vector pSUN5 (WO02 / 00900).
• fragment CHRC contains the CHRC promoter (1537 bp), fragment P-sense the 312 bp promoter fragment of epsilon cyclase in sense orientation, fragment intron the intron IV2 of the potato gene ST-LSI), and fragment P- anti the 312 bp promoter fragment of the epsilon cyclase in antisense orientation.
• fragment CHRC contains the CHRC promoter (1537 bp), fragment P-sense the 312 bp promoter fragment of epsilon cyclase in sense orientation, fragment intron the intron IV2 of the potato gene ST-LSI), fragment P-anti the 312 bp promoter fragment of the epsilon cyclase in antisense orientation and the fragment AP3P the 771 bp AP3P promoter fragment in antisense orientation.
• Example 6 Production of transgenic plants
• germination medium MS medium; Murashige and Skoog, Physiol. Plant. 15 (1962), 5 473-497) pH 5.8, 2% sucrose). Germination takes place in a temperature / light / time interval of 18 to 28 ° C / 20 to 200 for 4 to 8 weeks.
• the Agrobacterium tumefaciens strain EHA105 was treated with 'the binary plasmid pS5AI3 transformed. Culturing of the transformed A. tumefaciens strain EHA105 was grown overnight under the following conditions: A single colony was inoculated into YEB (0.1% yeast extract.
• the MS medium in which the leaves have been stored is replaced by the bacterial suspension
• the explants are cultivated for 1 to 8 days, but preferably for 6 days, the following conditions being able to be used: light intensity: 30 to 80 ocmol / m 2 x sec, temperature:
• the co-cultivated explants are then transferred to fresh MS medium, preferably with the same growth regulators, this second medium additionally containing an antibiotic to suppress bacterial growth.
• the explants are transferred to fresh medium until shoot buds and small shoots develop, which are then on the same basal medium including timentin and PPT or alternative components with growth regulators, namely, for example, 0.5 mg / 1 indolylbutyric acid (IBA) and 0.5 mg / 1 gibberillic acid GA 3 , are transferred for rooting. Rooted shoots can be transferred to the greenhouse.
• IBA indolylbutyric acid
• GA 3 gibberillic acid
• the explants. Before the explants. can be infected with the bacteria, +. NEN for 1 to 12 days, preferably 3 to 4, on the. medium for the co-culture described above are pre-incubated. The infection, co-culture and selective regeneration then take place as described above.
• Liquid culture medium can also be used for the entire process.
• the culture can also be incubated on commercially available carriers which are positioned on the liquid medium.
• the flower material of the transgenic Tagetes erecta plants from Example 6 was mortarized in liquid nitrogen and the powder (about 250 to 500 mg) extracted with 100% acetone (three times 500 ul each). The solvent was evaporated and the carotenoids resuspended in 100 ul acetone.
• the genetically modified plants Compared to the genetically unmodified control plant, the genetically modified plants have a significantly increased content of carotenoids of the " ⁇ -carotene pathway", such as, for example, ⁇ -carotene and zeaxanthin, and a significantly reduced content of carotenoids of the " ⁇ -carotene pathway” , such as lutein.
• ⁇ -carotene pathway such as, for example, ⁇ -carotene and zeaxanthin
• ⁇ -carotenoids of the " ⁇ -carotene pathway” such as lutein.
• Comparative Example 1 Reduction of the ⁇ -cyclase activity in Tagetes erecta by antisense

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