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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • 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
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  • 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

Definitions

• the present invention relates to novel genetic constructs such as expression cassettes and vectors for
• vitamin E tocopherol
• the eight naturally occurring compounds with vitamin E activity are derivatives of 6-chromanol (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft, Chapter 4., 478-488, Vitamin E).
• the first group (la-d) comprises the tocopherols (I)
• the second group (2a-d) comprises the tocopherols (II):
• R 1 , R 2 and R 3 are as defined above.
• Alpha-tocopherol is currently of greatest economic importance.
• a first subject of the invention therefore relates to an expression cassette containing, under genetic control, regulatory nucleic acid sequences
• HPPD 4-hydroxyphenyl pyruvate dioxygenase
• anti-HGD at least one nucleic acid sequence which is capable of inhibiting the homogentisate dioxygenase (HGD) activity.
• Inhibition or blocking of the HGD enzyme activity in the plant or the plant part or tissue transformed with an anti-HGD construct according to the invention also includes a quantitative reduction of active HGD in the plant up to an essentially complete absence (i.e. lack of detectability of HGD enzyme activity or lack of immunological detectability of HGD) of HGD protein.
• the strategy preferred according to the invention comprises the use of a nucleic acid sequence (anti-HGD) which can be transcribed to an antisense nucleic acid sequence which is capable of inhibiting the homogentisate dioxygenase (HGD) activity, e.g. B. by inhibiting the expression of endogenous HGD.
• anti-HGD a nucleic acid sequence
• HGD homogentisate dioxygenase
• An anti-HGD sequence in the sense of the present invention is therefore selected in particular from: a) antisense nucleic acid sequences; b) nucleic acid sequences coding for homologous HGD and leading to co-suppression c) viral nucleic acid sequences causing expression of HGD-RNA, and expression constructs; d) nonsense mutants of endogenous HGD-encoding nucleic acid sequences; e) nucleic acid sequences coding for knockout mutants; f) nucleic acid sequences suitable for homologous recombination; the expression of each of these sequences being able to "inhibit" the HGD activity in the sense of the invention.
• a combined application of such sequences is also conceivable.
• the coding HPPD sequence is preferably functionally linked to the coding sequence of a plant organ-specific transit peptide.
• the transit peptide preferably has specificity for the seeds or plastids, e.g. the plant's chloroplasts, chromoplasts and / or leukoplasts.
• the transit peptide directs the expressed HPPD activity to the desired target location in the plant and is preferably cleaved proteolytically from the HPPD protein part after it has been reached.
• the coding transit peptide sequence is preferably 5 'upstream of the coding HPPD sequence in the expression construct according to the invention.
• the coding HPPD sequence and the anti-HGD sequence are each under the genetic control of a plant-specific promoter.
• Expression cassettes which are particularly preferred according to the invention comprise a coding HPPD nucleic acid sequence which codes for a protein containing an amino acid sequence according to SEQ ID NO: 15 or a functional equivalent thereof, or the one Nucleic acid sequence from including nucleotide in position 8 to including nucleotide in position 1153 according to SEQ ID NO: 14 or a functional equivalent thereof.
• the anti-HGD nucleic acid sequence can contain the coding nucleic acid sequence of homogentisate dioxygenase inserted in the antisense orientation or a functional fragment thereof.
• a preferred embodiment of the expression cassettes according to the invention comprises an HGD sequence motif according to SEQ ID NO: 1 in an antisense orientation. This leads to the increased transcription of nucleic acid sequences in the transgenic plant which are complementary to the endogenous coding HGD sequence or a part thereof and hybridize with it at the DNA or RNA level.
• Another object of the invention relates to recombinant vectors comprising at least one expression cassette as defined above.
• vectors according to the invention include at least one expression construct of the following type:
• the coding HPPD sequence can also be replaced by a coding sequence for a fusion protein of transit peptide and HPPD.
• Preferred examples include monomeric vectors containing one of the following expression constructs:
• Constructs a) and b) require a co-transformation of the plant with both vectors, i.e. with a) and bl) or b2).
• Preferred examples also include binary vectors containing the following construct:
• Construct cl) or c2) allows the simultaneous transformation of the plant with HPPD and anti-HGD.
• Another object of the invention relates to microorganisms containing at least one recombinant vector according to the invention.
• Preferred organisms are those which are capable of infecting plants and thus of transmitting the constructs according to the invention.
• Preferred microorganisms are those from the genus Agrobacterium and in particular from the species Agrobacterium tumefaciens,
• Another object of the invention relates to the use of a vector or microorganism according to the invention for the transformation of plants, plant cells, plant tissues or parts, in particular with the aim of enabling them to improve tocopherol synthesis.
• Another object of the invention relates to transgenic plants transformed with at least one vector or microorganism according to the invention and transgenic cells, tissues, parts or transgenic reproductive material from such plants.
• transgenic plants according to the invention are selected in particular from crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potatoes, tobacco, tomato, rapeseed, alfalfa, lettuce, such as cress, and the various tree, Nut and wine species.
• crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potatoes, tobacco, tomato, rapeseed, alfalfa, lettuce, such as cress, and the various tree, Nut and wine species.
• the invention further relates to a process for the production of transgenic plants with improved tocopherol production, wherein plants which are capable of tocopherol production, or plant cells, tissue or parts or protoplasts thereof, are transformed with at least one vector according to the invention or at least one microorganism according to the invention transformed cells, tissues, parts of plants or protoplasts are cultivated in a growth medium and, if appropriate, plants are regenerated from the culture.
• Another object of the invention relates to the use of an expression cassette, a vector, a microorganism or a transgenic plant as defined above
• the last aspect of the invention relates to a process for the production of tocopherols, which is characterized in that one according to the invention is obtained from a culture transformed plant isolated the desired tocopherol in a manner known per se.
• a nucleotide or nucleic acid sequence is understood to mean, for example, a genomic or a complementary DNA sequence or an RNA sequence and semisynthetic or fully synthetic analogues thereof.
• HPPD or anti-HGD nucleotide sequences of the constructs according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components, and can consist of various heterologous HGD or HPPD gene segments from different organisms.
• the anti-HGD sequence can be derived from one or more exons and / or introns, in particular exons of the HGD gene.
• nucleotide sequences can be generated with codons, which are preferred by the plants to be transformed. These codons preferred by plants can be determined in the usual way for the plant on the basis of the codon usage.
• various DNA fragments can be manipulated in such a way that a nucleotide sequence with the correct reading direction and correct reading frame is obtained.
• adapters or linkers can be attached to the fragments.
• Functional equivalents of the HPPD gene are those sequences which, despite the differing nucleotide sequence, still code for a protein with the functions desired according to the invention, ie for an enzyme with homogentisate-forming activity.
• Functional equivalents of anti-HGD include those nucleotide sequences which sufficiently prevent the HGD enzyme function in the transgenic plant. This can be done, for example, by hindering or preventing HGD processing, the transport of HGD or its RNA, inhibition of ribosome attachment, inhibition of RNA splicing, induction of an RNA-degrading enzyme and / or inhibition of translation elongation or termination ,
• Functional equivalents include generally naturally occurring variants of the sequences described herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
• a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for HGD or HPPD, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
• the present invention also includes those nucleotide sequences which are obtained by modification of the HGD or HPPD nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme interfaces.
• Functional equivalents also include those variants whose function is weakened or enhanced compared to the starting gene or gene fragment, for example those HPPD genes which code for an HPPD variant with lower or higher enzymatic activity than that of the original gene.
• artificial nucleic acid sequences are suitable as long as, as described above, they impart the desired property, for example increasing the tocopherol content in the plant by overexpressing the HPPD gene or expressing an anti-HGD sequence in crop plants.
• Such artificial nucleotide sequences can be determined, for example, by back-translating proteins constructed using molecular modeling, which have HGD or HPPD activity, or by in vitro selection. Coding nucleotide sequences which are obtained by back-translating a polypeptide sequence in accordance with the codon usage specific for the host plant are particularly suitable. The specific codon usage can be determined by a person skilled in plant genetic methods Easily determine computer evaluations of other known genes of the plant to be transformed.
• DNA fragments can be retranslated, for example, based on the amino acid sequence of a bacterial HPPD and taking into account the plant codon usage, and the complete exogenous HPPD sequence optimized for use in the plant can be produced therefrom.
• An HPPD enzyme is expressed from this, which is not or only inadequately accessible to plant regulation, which means that the overexpression of enzyme activity can be fully exploited.
• suitable equivalent nucleic acid sequences are sequences which code for fusion proteins, part of the fusion protein being e.g. an HPPD polypeptide or a functionally equivalent part thereof.
• the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect HPPD expression (e.g. myc-tag or his-tag).
• this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the HPPD protein to the desired site of action.
• an increase in the tocopherol content in the plant means the artificially acquired ability of an increased biosynthetic capacity of at least one compound from the group of tocopherols and tocotrienols as defined above in the plant compared to the non-genetically modified plant for at least the duration a generation of plants.
• the tocopherol biosynthesis site is generally the leaf tissue but also the seed, so that leaf-specific and / or seed-specific expression, in particular of the HPPD gene and, if appropriate, of anti-HGD, is expedient.
• the tocopherol biosynthesis need not be limited to the seed, but can also be tissue-specific in all other parts of the plant.
• constitutive expression of the exogenous gene is advantageous.
• inducible expression may also be desirable.
• the regulatory nucleic acid sequences contained in the expression cassettes according to the invention optionally control the expression of the coding sequences (such as the HPPD sequence) fused with a transit peptide sequence) and the anti-HGD sequence.
• the constructs according to the invention preferably comprise a promoter 5 'upstream of the respective coding sequence and a terminator sequence 3' downstream and optionally further conventional regulative sequences
• An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence or the antisense sequence.
• sequences which can be linked operatively are further targeting sequences which differ from the transit peptide and which ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or others compartments; and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711), and the like.
• Suitable polyadenylation signals are plant polyadenylation signals, preferably those which essentially contain T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine syntha
• Ti plasmids correspond to pTiACHS (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents thereof.
• Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.
• any promoter that can control the expression of genes, in particular foreign genes, in plants is suitable as promoters for the expression cassettes.
• a plant promoter or a plant virus-derived promoter is preferably used.
• the CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980), 285-294).
• this promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).
• Another example of a suitable promoter is that of the LeguminB promoter (Accession No. X03677).
• the expression cassette can also contain a chemically inducible promoter, by means of which the expression of the exogenous gene in the plant can be controlled at a specific point in time.
• a chemically inducible promoter such as the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible (WO 95/19443), a benzenesulfonamide-inducible (EP- A-0388186), one which can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397404), one which can be induced by abscisic acid (EP-A 335528) or one which can be induced by ethanol or cyclohexanone (WO 93/21334) promoters can also be used.
• promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Promoters that ensure leaf-specific expression should be mentioned in particular.
• the promoter of the cytosolic FBPase from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989), 2445-245) are to be mentioned. Examples of seed-specific promoters are the
• Phaseolin promoter (US 5504200), the USP promoter (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3), 459-467) or the LEB4 promoter (Fiedler, U. et al., Biotechnology (NY) (1995), 13 (10) 1090) together with the LEB4 signal peptide.
• An expression cassette is produced by fusing a suitable promoter with a suitable anti-HDG or HPPD nucleotide sequence, optionally a sequence coding for a transit peptide, which is preferably arranged between the promoter and the HPPD sequence, and a terminator or polyadenylation signal , Common recombination and cloning techniques, such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.
• expression cassettes can also be used whose DNA sequence encodes an HPPD fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
• the following may be mentioned as examples: chloroplast-specific transit peptides, which after translocation of the HPPD gene into the chloroplasts from the HPPD part are enzymatically split off.
• this transit peptide e.g. the RubisCO small subunit transit peptide or the ferredoxin: NADP oxidoreductase.
• the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
• the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
• the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
• the promoter, terminator and the other regulatory elements can be both native (homologous) and foreign (heterologous) to the host plant.
• the expression cassettes according to the invention are preferably inserted into suitable transformation vectors.
• suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, pp. 71-119 (1993).
• agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as, for example. of tobacco plants can be used, for example, by wounded leaves or leaf pieces in one Agrobacteria solution are bathed and then cultivated in suitable media.
• the transformation of plants by agrobacteria is known, inter alia, from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press, 1993, pp. 15 - 38.
• Transgenic plants can be regenerated in a known manner from the transformed cells of the wounded leaves or leaf pieces.
• transformation The transfer of foreign genes into the genome of a plant is called transformation.
• the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called particle bombardment method, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by Agrobacterium.
• the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus, Annu. Rev. Plant Physiol.
• 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).
• Agrobacteria transformed with an expression cassette can also, in a known manner, for the transformation of plants, in particular crop plants, such as cereals, maize, oats, soybeans, rice, cotton, sugar beet, canola, sunflower, plachs, hemp, potatoes, tobacco, tomatoes, Rapeseed, alfalfa, lettuce and the various tree, nut and wine species can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
• crop plants such as cereals, maize, oats, soybeans, rice, cotton, sugar beet, canola, sunflower, plachs, hemp, potatoes, tobacco, tomatoes, Rapeseed, alfalfa, lettuce and the various tree, nut and wine species
• crop plants such as cereals, maize, oats, soybeans, rice, cotton, sugar beet, canola, sunflower, plachs, hemp, potatoes, tobacco, tomatoes, Rapeseed, alfalfa
• the invention also relates to transgenic plants transformed with an expression cassette according to the invention, and to transgenic cells, tissues, parts and propagation material of such plants.
• Transgenes are particularly preferred
• Cultivated plants such as barley, wheat, rye, corn, oats, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, Hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species.
• Plants in the sense of the invention are mono- and dicotyledonous plants or algae.
• Figure 1 is a schematic representation of the tocopherol biosynthetic pathway in plants
• PP stands for pyrophosphate
• geranyl-geranyl-PP in the plant (not shown), the corresponding tocotrienols are formed in an analogous manner
• FIG. 2 shows a binary transformation vector which expresses the HPPDop in seeds of transformed plants and at the same time suppresses the expression of the endogenous HGD:
• A 35S promoter;
• B HGD in antisense orientation;
• C OCS terminator;
• D Legumin B promoter;
• E transit peptide of the FNR;
• F HPPDop;
• G NOS terminator;
• FIG. 3 construction schemes of the plasmids pUC19HPPDop and pCRScriptHPPDop encoding HPPD;
• FIG. 4 construction schemes of the plasmids pBinARHGDanti and pCRScriptHGDanti encoding antiHGD;
• Figure 5 Construction schemes of the transformation vectors pPTVHGDanti and pPZP200HPPD.
• the sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from Licor (sold by MWG Biotech, Ebersbach) according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463 - 5467).
• Example 1 Cloning of a hydroxyphenyl pyruvate dioxygenase (HPPD) with a DNA sequence optimized for expression in Brassica napus
• the amino acid sequence of the hydroxyphenylpyruvate dioxygenase (HPPD) from Streptom ces averwitilis was translated back into a DNA sequence, taking into account the codon usage in Brassica napus (oilseed rape).
• the codon usage was determined using the database http://www.dna.affrc.go.jp/ -nakamura / index.html.
• the derived sequence was synthesized by attaching Sall interfaces by ligation of overlapping oligonucleotides with subsequent PCR amplification (Rouwendal, GJA; et al, (1997) PMB 33: 989-999) (SEQ ID NO: 14). The correctness of the sequence of the synthetic gene was checked by sequencing.
• the synthetic gene was cloned into the vector pBluescript II SK + (Stratagene).
• Open flowers were harvested from Brassica napus var. Westa and frozen in liquid nitrogen.
• the material was then pulverized in a mortar and taken up in Z6 buffer (8 M guanidinium hydrochloride, 20 mM MES, 20 mM EDTA, adjusted to pH 7.0 with NaOH; mixed with 400 ⁇ l mercaptoethanol / 100 ml buffer immediately before use) ,
• the suspension was then transferred to reaction vessels and shaken with a volume of phenol / chloroform / isoamyl alcohol 25: 24: 1.
• the supernatant was transferred to a new reaction vessel and the RNA was precipitated with 1/20 volume IN acetic acid and 0.7 volume ethanol (absolute).
• the pellet was first in 3M
• RNA 20 ⁇ g of total RNA were first mixed with 3.3 ⁇ l of 3M sodium acetate solution, 2 ⁇ l of IM magnesium sulfate solution and made up to a final volume of 10 ⁇ l with DEPC water. 1 ⁇ l of RNase-free DNase (Boehringer Mannheim) was added and incubated at 37 degrees for 45 min. After removing the enzyme by shaking with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was taken up in 100 ⁇ l DEPC water. 2.5 ⁇ g RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco BRL) according to the manufacturer's instructions.
• a cDNA kit Gibco BRL
• Oligonucleotides derived to which a Sall had been added at the 5 'end and an Asp718 restriction site had been added at the 3' end comprises the sequence:
• the oligonucleotide at the 3 'end comprises the sequence:
• GGTACCTCRAACATRAANGCCATNGTNCC SEQ ID NO: 3
• N in each case means inosine and R stands for the incorporation of A or G into the oligonucleotide.
• the PCR reaction was carried out with the Taq polymerase from TAKARA according to the manufacturer's instructions. 0.3 ⁇ g of the cDNA was used as template.
• the PCR program was:
• the fragment was purified using NucleoSpin Extract (Machery and Nagel) and cloned into the vector pGEMT (Promega) according to the manufacturer's instructions.
• the correctness of the fragment was checked by sequencing.
• Example 3 Production of a plant transformation construct for overexpression of the HPPD with an optimized DNA sequence (HPPDop) and switching off the HGD
• the components of the cassette for the expression of the HPPDop consisting of the LeguminB promoter (accession no. X03677), the transit peptide of ferredoxin: ADP + oxidoreductase from spinach (FNR; Jansen, T, et al (1988) Current Genetics 13, 517 -522) and the NOS terminator (contained in pBHOl Accession No. U12668) by means of PCR with the required restriction sites.
• legumin promoter was derived from the plasmid plePOCS (Bäumlein, H, et al. (1986) Plant J. 24, 233-239) with the upstream oligonucleotide:
• ATGGTACCTTTTTTGCATAAACTTATCTTCATAG (SEQ ID NO: 6)
• ATGTCGACCCGGGATCCAGGGCCCTGATGGGTCCCATTTTCCC SEQ ID NO: 7
• the NOS terminator was generated from the plasmid pBHOl (Jefferson, R.A., et al (1987) EMBO J. 6 (13), 3901-3907) by means of PCR with the 5 'oligonucleotide:
• AAGCTTCCGATCTAGTAACATAGA (SEQ ID NO: 9)
• the amplicon was cloned into the vector pCR-Script (Stratagene) according to the manufacturer's instructions.
• the NOS terminator was first cloned as a Sall / HindIII fragment into a correspondingly cut pUC19 vector (Yanisch-Perron, C, et al (1985) Gene 33, 103-119). The transit peptide was then introduced into this plasmid as an Asp718 / Sall fragment. The legumin promoter was then cloned in as an EcoRI / Asp718 fragment. The HPPDop gene was introduced into this construct as a Sall fragment (FIG. 3, construct III).
• the finished cassette in pUC19 was used as a template for a PCR, for which the oligonucleotide for the legume promoter:
• AAGCTTGATCTGTCGTCTCAAACTC (SEQ ID NO: 10)
• AAGCTTCCGATCTAGTAACATAGA (SEQ ID NO: 11) were used.
• the amplicon was cloned into pCR-Script and called pCR-ScriptHPPDop ( Figure 3, construct IV).
• the gene fragment was cloned as a Sall / Asp718 fragment into the vector pBinAR (Höfgen, R. and Willmitzer, L., (1990) Plant Sei. 66: 221-230), in which the 35S promoter and the OCS terminator 0 are present (FIG. 4, construct I).
• the construct served as a template for a PCR reaction with the oligonucleotide:
• the amplicon was cloned into the vector PCR script (Stratagene) and called HGDanti (FIG. 3, construct II).
• the construct HGDanti from pCRScriptHGDanti was first cloned as an Xbal fragment into the vector pPTV (Becker, D., (1992) PMB 20, 30 1195-1197) ( Figure 5, construct V).
• the construct LegHPPDop from pCRScriptHPPDop was inserted into this plasmid as a HindIII fragment. This plasmid was designated pPTVHPPD / HGDanti ( Figure 2, construct VI).
• transgenic oilseed rape plants were based on a protocol by Bade, J.B. and Damm, B. (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., ed., Springer Lab Manual, Springer Verlag, 1995, 30-38), in which also the composition of the media and buffers used is specified.
• the transformation was carried out with the Agrobacterium tumefaciens strain EHA105 (Li, X.Q., et al., PMB (1992) 20, 1037). Either the abovementioned plasmid 15 pPTVHPPDopHGDanti (FIG. 2) or, after cultivation, mixed cultures of agrobacteria with the plasmids pPTVHGDanti and pPZP200HPPDop (FIG. 5) were used for the transformation.
• Brassica napus var. Westar seeds were surface-sterilized with 70% ethanol (v / v) 20, washed in water for 10 minutes at 55 ° C, in 1% hypochlorite solution (25% v / v tea powder, 0.1% v / v Tween 20) incubated for 20 minutes and washed six times with sterile water for 20 minutes each.
• the seeds were dried on filter paper for three days and 10-15 seeds were germinated in a glass flask with 25 15 ml of germination medium.
• the roots and apices were removed from several seedlings (approx. 10 cm in size) and the remaining hypocotyls were cut into pieces approx. 6 mm long.
• the approximately 600 explants obtained in this way were washed with 50 ml of basal medium for 30 minutes and transferred to a 300 ml 30 flask. After adding 100 ml of callus induction medium, the cultures were incubated for 24 hours at 100 rpm.
• the callus induction medium was removed from the oilseed rape explants using sterile pipettes, 50 ml of Agrobacterium solution 45 were added, mixed carefully and incubated for 20 min.
• Petri dishes transferred containing 25 ml of shoot induction medium with phosphinotricin.
• the petri dishes were closed with 2 layers of leucopor and incubated at 25 ° C. and 2000 lux with photoperiods of 16 hours light / 8 hours dark.
• the developing calli were transferred to fresh petri dishes with shoot induction medium every 12 days. All further steps for the regeneration of whole plants were carried out as by Bade, JB and Damm, B. (in: Gene Transfer to Plants, Potrykus, I. and Spangenberg, G., ed., Springer Lab Manual, Springer Verlag, 1995, 30- 38).

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