EP1880011A2 - Cassettes d'expression transgenique utilisees pour l'expression d'acides nucleiques dans des tissus floraux de plantes - Google Patents

Cassettes d'expression transgenique utilisees pour l'expression d'acides nucleiques dans des tissus floraux de plantes

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Publication number
EP1880011A2
EP1880011A2 EP06754978A EP06754978A EP1880011A2 EP 1880011 A2 EP1880011 A2 EP 1880011A2 EP 06754978 A EP06754978 A EP 06754978A EP 06754978 A EP06754978 A EP 06754978A EP 1880011 A2 EP1880011 A2 EP 1880011A2
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
promoter
acid sequence
sequences
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EP06754978A
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German (de)
English (en)
Inventor
Christel Renate Schopfer
Matt Sauer
Ralf Flachmann
Martin Klebsattel
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SunGene GmbH
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SunGene GmbH
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Publication of EP1880011A2 publication Critical patent/EP1880011A2/fr
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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
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention relates to methods for targeted, transgenic expression of nucleic acid sequences in flower tissues of plants, as well as transgenic expression cassettes and expression vectors containing promoters with expression specificity for tissue of the flower.
  • the invention further relates to organisms transformed with these transgenic expression cassettes or expression vectors (preferably plants), cultures, parts or propagation material derived therefrom, and the use thereof for the production of foodstuffs, feedstuffs, seeds, pharmaceuticals or fine chemicals.
  • the aim of biotechnological work on plants is the production of plants with advantageous new properties, for example, to increase agricultural productivity, improve the quality of food or to produce certain chemicals or pharmaceuticals (Dunwell JM (2000) J Exp Bot 51 Spec No: 487-96 ).
  • a prerequisite for the transgenic expression of certain genes is the provision of plant-specific promoters. Promoters are important
  • constitutive promoters such as the Agrobacterium nopaline synthase promoter, the TR double promoter, or the cauliflower mosaic virus 35S transcript (CaMV) promoter (Odell et al., (1985) Nature 313: 810-812).
  • CaMV cauliflower mosaic virus 35S transcript
  • promoters with specificities for various plant tissues such as anthers, ovaries, flowers, leaves, stems, roots, tubers or seeds.
  • the stringency of specificity, as well as the expression activity of these promoters is very different.
  • the plant bloom serves the sexual reproduction of the seed plants.
  • phytochemicals such as terpenes, anthocyanins, carotenoids, alkaloids and phenylpropanoids, which are used as fragrances, antibodies or as dyes. Many of these substances are of economic interest.
  • To- The flower bud and the flower of the plant is a sensitive organ, especially against stress factors like cold.
  • the Arabidopsis thaliana gene locus At5g33370 (derived protein GenBank Acc No .: NP_198322) encodes a putative GDSL motif lipase / hydrolase family protein.
  • the Arabidopsis thaliana gene locus At5g22430 (derived protein GenBank Acc No .: NP_568418) encodes an expressed protein.
  • the Arabidopsis thaliana gene locus At1g26630 (derived protein GenBank Acc. No .: NP_173985) encodes a putative eukaryotic translation initiation factor 5A / elF-5.
  • the Arabidopsis thaliana gene locus At4g35100 (derived protein GenBank Acc. No .: NP_195236) encodes a putative plasma membrane intrinsic protein (SIMIP).
  • the Arabidopsis thaliana gene locus At3g04290 (derived protein GenBank Acc.
  • NP_187079 encodes a putative GDSL-motif lipase / hydrolase family protein.
  • the Arabidopsis thaliana gene locus At5g46110 (derived protein GenBank Acc. No .: NP_568655) encodes a putative phosphate / triose-phosphate translocator.
  • a first subject of the invention relates to methods for the targeted, transgenic expression of nucleic acid sequences in flower tissues of plants, the following steps being included:
  • transgenic expression cassette contains at least the following elements
  • transgenic cells containing said expression cassette stably integrated into the genome and IN THE. Regeneration of whole plants from said transgenic cells, wherein at least one of the further nucleic acid sequence is expressed in substantially all flower tissues.
  • transgenic expression cassettes such as e.g. can be used in the method according to the invention.
  • the transgenic expression cassettes comprise for targeted, transgenic expression of nucleic acid sequences in flower tissues of plants,
  • the expression cassettes according to the invention may contain further genetic control sequences and / or additional functional elements.
  • the transgenic expression cassettes can preferably be expressed by the transgenic nucleic acid sequence expressing a protein encoded by said nucleic acid sequence, and / or expressing one of said nucleic acid sequences. sequence-encoded sense RNA, anti-sense RNA or double-stranded RNA.
  • transgenic expression cassettes according to the invention are particularly advantageous because they provide selective expression in the tissues of the flower bud and the flower of the plant and allow numerous applications, such as resistance to stress factors such as cold or targeted synthesis of phytochemicals.
  • the expression is essentially constant over the entire developmental period of the flower bud and flower.
  • transgenic expression cassettes according to the invention, the transgenic expression vectors and transgenic organisms derived therefrom can functional equivalents to the promoter sequences described under SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 include.
  • Another object of the invention relates to transgenic expression vectors containing one of the expression cassettes of the invention.
  • Another object of the invention relates to transgenic organisms containing one of the expression cassettes or expression vectors of the invention.
  • the organism may be selected from the group consisting of bacteria, yeasts, fungi, non-human animal and plant organisms or derived cells, cell cultures, parts, tissues, organs or propagation material, preferably the organism is selected from the group of agricultural uses - plants.
  • Another object of the invention relates to the use of said organisms or cells derived therefrom, cell cultures, parts, tissues, organs or propagation material for the production of food, feed, seeds, pharmaceuticals or fine chemicals, the fine chemicals preferably enzymes, vitamins, amino acids, Sugar, saturated or unsaturated fatty acids, natural or synthetic flavorings, flavorings or colorings. Also included according to the invention are processes for producing said foods, feedstuffs, seeds, pharmaceuticals or fine chemicals using the organisms according to the invention or cells, cell cultures, parts, tissues, organs or propagation material derived therefrom.
  • the promoter activity of a functionally equivalent promoter is termed “essentially the same” if the transcription of a particular transgenic nucleic acid sequence under control of the said functionally equivalent promoter under otherwise unchanged conditions shows targeted expression in essentially all flower tissues.
  • “Flower” generally means a shoot of limited growth, the leaves of which have been transformed into reproductive organs.
  • the flower consists of various "flower tissues” such as the sepals (Sepalen), the petals, the stamens (or dust “vessels", Stamina) or the carpels (carpels).
  • the androeceum in the flower is called the whole of the stamina.
  • the stamens are located within the petal or Sepalenkieris.
  • a stamen is divided into a filament and an anther sitting at the end. This in turn is divided into two counters, which are connected by a connective. Each counter consists of two pollen sacs in which the pollen is formed.
  • Substantially all flower tissue means, with respect to the flower tissues, that some of these tissues may not exhibit substantial expression as a whole or at particular times of development, but the proportion of these tissues is preferably less than 20% by weight, preferably less than 10%. %, more preferably less than 5% by weight, most preferably less than 1% by weight of the total weight of the flower tissues.
  • “Targeted” with respect to the expression in flower tissues preferably means that the expression under control of one of the promoters according to the invention in the flower tissues is preferably at least twice, more preferably at least ten times, most preferably at least a hundred times higher than in a non -Blossom tissue such as the leaves.
  • the promoters according to the invention "show substantially no expression in the pollen and ovaries" preferably means that the statistical mean value of the expression over all reproductive floral tissue is at most 10%, preferably at most 5%, most preferably at most 1% of the statistical average of Expression over all flower tissues under the same conditions.
  • the expression within the flower tissue is substantially constant.
  • “Substantially constant” means preferably that the standard deviation of the expression between the individual flower tissues based on the statistical average expression over all flower tissue is less than 50%, preferably 20%, more preferably 10%, most preferably 5%.
  • the expression within at least one particular flower tissue is substantially constant throughout all stages of development of the flower.
  • “Substantially constant” here preferably means that the standard deviation of the expression between the individual development times of the respective flower tissue based on the statistical average expression over all development times is less than 50%, preferably 20%, particularly preferably 10%, very particularly preferably 5 %.
  • preference is given to using those nucleic acid sequences in functional linkage with the promoter to be tested which code for readily quantifiable proteins.
  • reporter proteins Schoenborn E, Groskreutz D (1999) Mol Biotechnol 13 (1): 29-44) such as "green fluorescence protein” (GFP) (Chui WL et al.
  • “Otherwise unchanged conditions” means that the expression initiated by one of the transgenic expression cassettes to be compared is not modified by combination with additional genetic control sequences, for example enhancer sequences. Unchanged conditions also means that all framework conditions such as plant species, developmental stage of the plants, culture conditions, assay conditions (such as buffer, temperature, substrates, etc.) are kept identical between the expressions to be compared.
  • transgene it is meant, for example, with respect to an expression cassette, or expression vector or transgenic organism comprising it, all such constructions made by genetic engineering in which either
  • the promoter sequence according to the invention contained in the expression cassettes is preferably heterologous with respect to FIG their functionally linked, transgene-expressing further Nukleinklaresequzenz.
  • heterologous in this context means that the further nucleic acid sequence does not encode the gene that is naturally under the control of said promoter.
  • Natural genetic environment means the natural chromosomal locus in the lineage or the presence in a genomic library.
  • the natural, genetic environment of the nucleic acid sequence is preferably at least partially preserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, more preferably at least 1000 bp, most preferably at least 5000 bp.
  • a naturally occurring expression cassette for example the naturally occurring combination of the promoter of a gene coding for a protein according to the genes with the gene-localizations At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110 or a functional equivalent thereof with its corresponding coding sequences becomes a transgenic expression construct when modified by non-natural, synthetic ("artificial") methods such as mutagenization.
  • non-natural, synthetic (“artificial") methods such as mutagenization.
  • Transgene in terms of expression preferably means all those realized using a transgenic expression cassette, a transgenic expression vector or transgenic organism - according to the definitions given above - realized expressions.
  • “Functional equivalents" of a promoter according to SEQ ID NO: 1, 4, 7, 10, 11 and 12 means in particular natural or artificial mutations of a promoter, for example according to SEQ ID NO: 2, 3, 5, 6, 8, and 9, and homologous sequences from other organisms, preferably from plant organisms, which have essentially the same promoter activity as one of the promoters according to SEQ ID NO: 1, 4, 7, 10, 11 or 12.
  • Functional equivalents also include all of the sequences derived from the complementary complementary strand of the sequences defined by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively have the same promoter activity.
  • a) have substantially the same promoter activity as one of the promoters according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 respectively and
  • the expression level of the functional equivalents may differ both downwards and upwards compared to a comparison value. Preference is given to those sequences whose expression level, measured on the basis of the transcribed mRNA or as a result translated protein, under otherwise unchanged conditions quantitatively by not more than 50%, preferably 25%, particularly preferably 10% obtained from a comparison value with those by SEQ ID NO : 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 described promoters.
  • Examples of the promoter sequences used in the transgenic expression cassettes or transgenic expression vectors according to the invention can be found, for example, in other organisms whose genomic sequence is known, such as Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthium annuus, Linum sativum by homology comparisons Easily find databases. For this purpose, preference can be given to starting from the coding regions of the genes whose promoters are described for example by SEQ ID NO 1, 4, 7, 10, 11 or 12.
  • the sequences of the genes with the gene site names At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110 the corresponding homologous genes in other plant species by screening databases or gene banks (using appropriate gene probes) easily in the specialist common way to be identified.
  • functional equivalents of the promoters described by SEQ ID NO: 1, 4, 7, 10, 11 and 12 include all those promoters which are in a 5'-direction in a plant organism before a genomic Sequence which code for a protein having a homology of at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%.
  • these are the genes with the gene cluster names At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110 corresponding to the proteins with the sequences according to Acc. No.
  • a nucleic acid sequence for example a gene transcript such as a cDNA.
  • all methods for the amplification of flanking chromosomal sequences are available.
  • the two most commonly used methods are the inverse PCR ("iPCR", shown schematically in Figure 10) and the "Thermal Asymmetry Interlaced PCR"("TAILPCR”).
  • genomic DNA of the organism from which the functionally equivalent promoter is to be isolated is completely digested with a given restriction enzyme, and then the individual fragments are back ligated in a dilute approach, that is, combined with themselves into an annular molecule.
  • the large number of ring-shaped DNA molecules present also contains those which contain the known sequence (for example the sequence coding for the homologous protein). Proceeding from this, the circular molecule can be amplified by PCR using a primer pair in which both primers can anneal to the known sequence segment.
  • One possible embodiment for the "iPCR” is shown by way of example in Example 4.
  • the "TAIL-PCR” is based on the use of a set of successively shortened highly specific primers which attach to the known genomic sequence (for example the sequence coding for the homologous protein) and on the other hand a set of shorter, low-melting-point random primers a sequence-unspecific attachment to the known genomic sequence flanking genomic DNA is carried out.
  • the attachment of the primer to the DNA to be amplified can be designed with such a primer combination so that a specific amplification of the desired target sequence is possible.
  • One possible embodiment for the "TAIL-PCR” is reproduced by way of example in Example 4.
  • Another object of the invention relates to processes for the preparation of a transgene expression cassette with specificity for flower tissue, comprising the following steps:
  • Isolation of a promoter with specificity for flower tissue wherein in the isolation at least one nucleic acid sequence or a part thereof is used, wherein said nucleic acid sequence encodes an amino acid sequence comprising at least part of the sequences according to the Acc. No. NP_198322, NP_568418, NP_173985, NP_195236, NP_187079 or NP_568655;
  • said nucleic acid sequence encodes an amino acid sequence comprising sequences according to Acc. No. NP_198322, NP_568418, NP_173985, NP_195236, NP_187079 or NP_568655.
  • Part with reference to the nucleic acid sequence preferably means a sequence of at least 10 bases, preferably 15 bases, more preferably 20 bases, most preferably 30 bases.
  • the method according to the invention is based on the polymerase chain reaction, wherein the said nucleic acid sequence or a part thereof is used as primer.
  • methods known to those skilled in the art such as e.g. Ligation, etc. are used (s.u.).
  • “Mutation” means substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • those nucleotide sequences are also encompassed by the present invention, which are obtained by modification of the promoters according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively receives.
  • the aim of such modification may be to further confine the sequence contained therein or e.g. also the insertion of further restriction enzyme cleavage sites, the removal of superfluous DNA or the addition of further sequences, for example further regulatory sequences.
  • Transitions and transversions may be used, techniques known per se, such as in vitro mutagenesis, primer repair, restriction or ligation may be used.
  • Transition means a base pair exchange of one purine / pyrimidine pair into another purine / pyrimidine pair (e.g., A-T versus G-C).
  • Transversion means a base pair exchange of a purine / pyrimidine pair for a pyrimidine / purine pair (e.g., A-T for T-A).
  • Deletion means the removal of one or more base pairs.
  • Insertion means the introduction of one or more base pairs.
  • identity between two nucleic acids is meant the identity of the nucleotides over the respective total length of the nucleic acid, in particular the identity of the nucleic acids Comparison using the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM, Fast and sensitive multiple sequence alignments on a microcomputer, Comput Appl. Biosci, 1989 Apr; 5 (2): 151-1) is calculated by setting the following parameters:
  • Identity between two proteins is understood to mean the identity of the amino acids over the entire protein length, in particular the identity determined by comparison with the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM and sensitive multiple sequence alignments on a microcomputer, Comput Appl. Biosci 1989 Apr; 5 (2): 151-1) is calculated with the following parameters:
  • Gap Separation penalty ranks 8 Gap separation penalty off
  • Gap Weight 8 Length Weight: 2
  • a sequence which has a homology of at least 60% on a protein basis with the sequences according to NP_198322, NP_568418, NP_173985, NPJI95236, NP_187079, NP_568655 a sequence understood that in a comparison with the above program algorithm with the above parameter set a homology of at least 60%.
  • Functional equivalents also mean DNA sequences which under standard conditions with one of the nucleic acid sequences coding for one of the promoters according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or the hybridize to these complementary nucleic acid sequences and have substantially the same promoter properties.
  • Standard hybridization conditions is to be understood broadly and means both stringent and less stringent hybridization conditions.
  • Such hybridization conditions are described, inter alia, in Sambrook J, Fritsch EF, Maniatis T et al., In Molecular Cloning - A Laboratory Manual, 2nd Ed., CoId Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57 or in Current Protocols in Molecular Biolo- gy, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. described.
  • the Conditions during the washing step should be selected from the range of conditions limited by those of low stringency (with about 2X SSC at 50 ° C) and those with high stringency (with about 0.2X SSC at 50 ° C preferably at 65 ° C) (2OX SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0).
  • the temperature during the washing step can be raised from low stringency conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied at the same time, also one of the two parameters can be kept constant and only the other can be varied.
  • denicating agents such as, for example, formamide or SDS. In the presence of 50% formamide, hybridization is preferably carried out at 42 ° C.
  • Methods for the production of functional equivalents according to the invention preferably include the introduction of mutations into one of the promoters according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively. Mutagenesis may be undirected
  • the mutagenized sequences are then screened for their properties according to a "trial-and-error" procedure. Particularly advantageous selection criteria include, for example, the level of the resulting expression of the introduced nucleic acid sequence in a flower tissue.
  • Methods for the mutagenization of nucleic acid sequences are known to the person skilled in the art and include, for example, the use of oligonucleotides having one or more mutations in comparison to the region to be mutated (eg in the context of a "site-specific mutagenesis").
  • primers of about 15 to about 75 nucleotides or more are employed, with preferably about 10 to about 25 or more nucleotide residues located on either side of the sequence to be altered. Details and implementation of said mutagenesis procedures are well known to those skilled in the art (Kunkel et al., (1987) Methods Enzymol 154: 367-382, Tomic et al (1990) Nucl Acids Res 12: 1656, Upender et al (1995) Biotechniques 18 (1 ): 29-30; US 4,237,224). Mutagenesis may also be accomplished by treating, for example, transgenic expression vectors containing one of the nucleic acid sequences of the invention with mutagenizing agents such as hydroxylamine.
  • non-essential sequences of a promoter according to the invention can be deleted without significantly impairing the abovementioned essential properties.
  • deletion variants represent functional equivalents to the promoters described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively, or to functional equivalents thereof.
  • the limitation of Promoter sequence to specific, essential regulatory regions can eg be made with the help of search engine for the search of promoter elements. Often, certain promoter elements are abundant in the regions relevant to promoter activity.
  • the functionally equivalent fragments comprise one of the promoters according to the invention - for example the promoters described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 - at least 200 base pairs, completely more preferably at least 500 base pairs, most preferably at least 1000 base pairs of the 3 'end of the respective promoter according to the invention - for example the promoters described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12 -, the length being calculated from the transcription start ("ATG" codon) in the 5'-direction upstream.
  • Very particularly preferred functional equivalents are the promoter sequences described by SEQ ID NO: 2, 3, 5, 6, 8 or 9.
  • Further functionally equivalent fragments can be generated, for example, by deletion of possibly existing 5'-untranslated regions. For this purpose, the
  • ren such as 5'-RACE
  • ren are determined and the 5'-untranslated be deleted by PCR-mediated methods or Endonukleaseverdau.
  • At least one of the promoters according to the invention is in functional linkage with at least one transgenic to be expressed nucleic acid sequence.
  • a functional linkage is understood as meaning, for example, the sequential arrangement of one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). with a nucleic acid sequence to be expressed transgenically and optionally further genetic control sequences such as a terminator or a polyadenylation sequence such that the promoter can fulfill its function in transgenic expression of the nucleic acid sequence under suitable conditions and the expression of the nucleic acid sequence (ie transcription and optionally Translation).
  • Suitable conditions mean preferably the presence of the expression cassette in a plant cell, preferably a plant cell comprised of a flower tissue of a plant.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, more preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
  • transgenic expression construct consisting of a linkage of promoter and nucleic acid sequence to be expressed, can be present integrated in a vector and inserted by, for example, transformation into a plant genome.
  • an expression cassette is also to be understood as those constructions in which one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) without it having been previously necessarily functionally linked to a nucleic acid sequence to be expressed, introduced, for example, via a targeted homologous recombination or a random insertion into a host genome, where it assumes regulatory control over endogenous nucleic acid sequences operatively linked thereto and controls the transgenic expression thereof , Insertion of the promoter-for example by homologous recombination-in front of a nucleic acid coding for a specific polypeptide results in an expression cassette according to the invention which controls the expression of the particular polypeptide selectively in the tissues of the flower.
  • the natural promoter of an endogenous gene can be exchanged for one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) and so on the expression behavior of the endogenous gene can be modified.
  • the insertion of the promoter may also be such that antisense RNA is expressed to the nucleic acid encoding a particular polypeptide.
  • antisense RNA is expressed to the nucleic acid encoding a particular polypeptide.
  • nucleic acid sequence to be expressed transgenically - for example by a homologous recombination - downstream of the sequence coding for one of the promoters of the invention eg described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11 and 12, respectively
  • an expression cassette according to the invention which controls the expression of the transgenic nucleic acid sequence to be expressed in the flower tissues.
  • the transgenic expression cassettes according to the invention may comprise further genetic control sequences.
  • the term "genetic control sequences" is to be understood broadly and means all those sequences which have an influence on the occurrence or the function of a transgenic expression cassette according to the invention. Genetic control sequences, for example, modify transcription and translation in prokaryotic or eukaryotic organisms.
  • the transgenic expression cassettes according to the invention 3'-downstream of the respective transgenic nucleic acid sequence comprise a terminator sequence as an additional genetic control sequence, and optionally further customary regulatory elements, in each case functionally linked to the transgene to be expressed nucleic acid sequence.
  • Genetic control sequences also include other promoters, promoter elements or mini-promoters that can modify the expression-controlling properties.
  • the tissue-specific expression can additionally take place as a function of specific stress factors.
  • Corresponding elements are described, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217 (2). 3): 246-53).
  • promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable transgenic expression in further plant tissues or in other organisms, such as, for example, E. coli bacteria.
  • Suitable promoters are in principle all plant-specific promoters in question.
  • Plant-specific promoters basically means any promoter that can control the expression of genes, especially foreign genes, in plants or plant parts, cells, tissues, cultures.
  • the expression may be, for example, constitutive, inducible or developmentally dependent.
  • Corresponding promoters are generally known to the person skilled in the art.
  • control sequences can be found, for example, in the promoters of Gram-positive bacteria such as amy and SPO2 or in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • Genetic control sequences also include the 5 'untranslated regions, introns or non-coding 3' region of genes such as the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (commonly: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)), preferably the genes having the gene locus At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110 from Arabidopsis thaliana. It can be shown that such regions can play a significant role in the regulation of gene expression. It has been shown that 5'-untranslated sequences can enhance the transient expression of heterologous genes.
  • Exemplary of translation enhancers is the 5'-leader sequence from the tobacco mosaic virus (GaIMe et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They may also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).
  • the nucleic acid sequences given under SEQ ID NO: 1, 4, 7, 10, 11 and 12 respectively represent the promoter region and the 5'-untranslated regions up to the ATG start codon of the respective ones Genes with the gene locus At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110.
  • the transgenic expression construct may advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which allow increased transgene expression of the nucleic acid sequence. Also at the 3 'end of the transgenic nucleic acid sequences to be expressed additional advantageous sequences can be inserted, such as other regulatory elements or terminators.
  • the transgenic nucleic acid sequences to be expressed can be contained in one or more copies in the gene construct.
  • Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which are essentially T-DNA polyadenylation signals from Agrobacterium tumefaciens.
  • Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator.
  • Control sequences are furthermore to be understood as meaning those which permit homologous recombination or insertion into the genome of a host organism or permit removal from the genome.
  • the coding sequence of a particular endogenous gene can be selectively exchanged for the sequence coding for a dsRNA.
  • Methods such as the cre / lox technology allow a tissue-specific, possibly inducible removal of the transgenic expression construct from the genome of the host organism (Sauer B (1998) Methods 14 (4): 381-92).
  • certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.
  • a transgenic expression cassette and / or the transgenic expression vectors derived therefrom may contain further functional elements.
  • the term functional element is to be understood broadly and means all those elements which have an influence on the production, propagation or function of the transgenic expression constructs according to the invention, the transgenic expression vectors or the transgenic organisms.
  • metabolism inhibitors eg 2-deoxyglucose-6-phosphate, WO 98/45456
  • antibiotics eg kanamycin, G 418, bleomycin, hygromycin
  • - preferably - herbicides eg phosphinotriquin
  • selection markers are: phosphinothricin acetyltransferases (bar and pat gene) which inactivate glutamine synthase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthases (EPSP synthase genes) which confer resistance to glyphosatr (N- (phosphonomethyl) glycine), Glyphosate degrading enzymes (gox gene product, glyphosate oxidoreductase), dehalogenases which, for example, inactivate dalapone (deh gene product), sulfonylurea and imidazolinone inactivating acetolactate synthases and nitrilases, which degrade eg bromoxynil (bxn gene product), the aasa gene product, which is a resistance against the antibiotic apectinomycin distribution, streptomycin phosphotransferases (SPT) that confer resistance to streptomycin, neomycin
  • Variants with e.g. the S4 and / or Hra mutation).
  • Reporter genes which code for easily quantifiable proteins and ensure an evaluation of the transformation efficiency or of the expression site or time point via intrinsic color or enzyme activity. Very particular preference is given to reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol 1999, 13 (1): 29-44), such as the "green fluorescence protein” (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784), chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859), the aequorin gene (Prasher et al.
  • GFP green fluorescence protein
  • origins of replication comprising an increase of the transgenic expression constructs or transgenic expression vectors according to the invention in, for example,
  • E.coli examples include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sambrook et al .: Molecular Cloning, A Laboratory Manual, 2 nd ed., CoId Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989 ).
  • “Introduction” in the context of the invention encompasses all methods which are suitable for directly or indirectly, a nucleic acid sequence (for example an expression cassette according to the invention), into an organism (eg a plant) or a cell, association, tissue, organ or propagation material ( Eg seeds or FR réelle) introduce the same or generate there. Direct and indirect procedures are included. The introduction can lead to a transient (transient) presence of said nucleic acid sequence or else to a permanent (stable) one. Introduction includes, for example, methods such as transfection, transduction or transformation. The organisms used in the process are grown or bred, depending on the host organism, in a manner known to those skilled in the art.
  • transgenic expression cassettes according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably in plants or plant cells, tissues, organs, parts or seeds) can advantageously be realized using vectors in which the transgenic expression cassettes are included.
  • Vectors may be, for example, plasmids, cosmids, phages, viruses or agrobacteria.
  • the transgenic expression cassettes can be inserted into the vector (preferably a plasmid vector) via a suitable restriction site.
  • the resulting vector can first be introduced into E. coli and amplified. Correctly transformed E. coli are selected, grown and the recombinant vector obtained by methods familiar to those skilled in the art. Restriction analysis and sequencing can serve to verify the cloning step. Preference is given to those vectors which enable a stable integration of the expression cassette into the host genome.
  • a transformed organism or a transformed cell or tissue
  • the appropriate DNA e.g., the expression vector
  • RNA be introduced into the appropriate host cell.
  • transformation or transfection
  • the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can be permeabilized chemically, for example with polyethylene glycol, so that the DNA can enter the cell by diffusion.
  • the DNA may also be made by protoplast fusion with other DNA-containing moieties such as minicells, cells, lysosomes or liposomes.
  • Electroporation is another suitable method for introducing DNA, in which the cells are reversibly permeabilized by an electrical pulse.
  • Corresponding methods have been described (for example, Bilang et al., (1991) Gene 100: 247-250, Scheid et al (1991) Mol Gen Gen 228: 104-112, Guerche et al (1987) Plant Science 52: 111- Neuhause et al., (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73; Howell et al. (1980) Science 208: 1265; Horsch et al. (1985 ) Science 227: 1229-1231; DeBlock et al.
  • vectors for expression in E. coli preferred are pQE70, pQE60 and pQE-9 (QIAGEN, Inc.); pBluescript vectors, phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).
  • Preferred vectors for expression in mammalian cells include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG and pSVL (Pharmacia Biotech, Inc.).
  • inducible vectors are pTet-tTak, pTet-splice, pcDNA4 / TO, pcDNA4 / TO / LacZ, pcDNA6 / TR, pcDNA4fl " O / Myc-His / LacZ, pcDNA4 / TO / Myc-His A, pcDNA4flO / Myc-His B, pcDNA4flO / Myc-His C, pVgRXR (Invitrogen, Inc.), or the pMAM series (Clontech, Inc., GenBank Accession No .: U02443), which already provide the inducible regulatory control element for, e.g., chemically, inducible expression to disposal.
  • Vectors for expression in yeast include, by way of example, pYES2, pYD1, pTEFI / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2, PHIL-SI, pPIC3SK, pPIC9K, and PA0815 (Invitrogen, Inc .).
  • Cloning vectors and techniques for the genetic manipulation of ciliates and algae are known to the person skilled in the art (WO 98/01572; Falciatore et al. (1999) Marine Biotechnology 1 (3): 239-251; Dunahay et al. (1995) J Phycol 31 : from 10,004 to 1,012).
  • Suitable methods are, in particular, protoplast transformation by polyethylene glycol-induced DNA uptake, calcium phosphate-mediated transformation, DEAE-dextran-mediated transformation, liposome-mediated transformation (Freeman et al., (1984) Plant Cell Physiol 29: 1353 et seq., US 4,536,475), biolistic methods using the particle bombardment method (US 5,100,792; EP-A 0 444 882; EP-A-0 434 616; Fromm ME et al. (1990) Bio / Technology 8 (9): 833-9; Gordon -Kamm et al.
  • transformation may also be by bacterial infection by Agrobacterium (eg EP 0 116 718), viral infection by viral vectors (EP 0 067 553, US 4,407,956, WO 95/34668, WO 93/03161) or by pollen (EP 0 270 356, WO 85/01856, US 4,684,611).
  • Agrobacterium eg EP 0 116 718
  • viral infection by viral vectors
  • pollen EP 0 270 356, WO 85/01856, US 4,684,611
  • the transformation is preferably carried out by means of agrobacteria which contain "disarmed" Ti plasmid vectors, the natural ability of which is utilized for gene transfer to plants (EP-A 0 270 355, EP-A 0 116 718).
  • Agrobacterium transformation is widely used for the transformation of dicotyledons, but is also increasingly applied to monocotyledons (Toriyama et al., (1988) Bio / Technology 6: 1072-1074; Zhang et al. (1988) Plant Cell Rep. 379-384; Zhang et al. (1988) Theor Appl Genet 76: 835-840; Shimamoto et al. (1989) Nature 338: 274-276; Datta et al. (1990) Bio / Technology 8: 736-740; Christou et al. (1991) Bio / Technology 9: 957-962; Peng et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao et al.
  • the strains Agrobacterium tumefaciens or Agrobacterium rhizogenes most commonly used for the Agrobacterium transformation contain a plasmid (Ti or Ri plasmid) which is transferred to the plant after Agrobacterium infection. Part of this plasmid, called T-DNA (transferred DNA), is integrated into the genome of the plant cell.
  • Agrobacterium also allows binary vectors (mini-Ti plasmids) to be transferred to plants and integrated into their genome.
  • Agrobacterium tumefaciens for the transformation of plants using tissue culture explants has been described (et al., Horsch RB et al., (1985) Science 225: 1229ff; Fraley et al. (1983) Proc Natl Acad. See USA 80: 4803-4807; Bevans et al. (1983) Nature 304: 184-187).
  • strains of Agrobacterium tumefaciens are able to transfer genetic material, for example the expression cassettes according to the invention, such as the strains EHA101 [pEHA101], EHA105 [pEHA105], LBA4404 [pAL4404], C58C1 [pMP90] and C58C1 [pGV2260] ( Hood et al (1993) Transgenic Res 2: 208-218; Hoekema et al. (1983) Nature 303: 179-181; Koncz and Schell (1986) Gene Genet 204: 383-396; Debreche et al. 1985) Nucl Acids Res 13: 4777-4788).
  • the expression cassette is to be integrated into special plasmids, either into an intermediate vector (shuttle or intermediate vector) or a binary vector.
  • binary vectors which can replicate both in E. coli and in Agrobacterium. They usually contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA restriction sequences. They can be transformed directly into Agrobacterium (Holsters et al., (1978) Mol Gen Genet 163: 181-187).
  • the Agrobacterium acting as host organism in this case should already contain a plasmid with the vir region. This is required for the transfer of T-DNA to the plant cell.
  • Such transformed Agrobacterium can be used to transform plant cells.
  • T-DNA T-DNA to transform plant cells has been extensively studied and described (EP-A-0 120 516, Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V, An et al., (1985) EMBO J 4: 277-287).
  • Various binary vectors are known and are partially available commercially, such as pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res 12: 8711), pBinAR, pPZP200 or pPTV.
  • the agrobacteria transformed with such a vector can then be used in a known manner for the transformation of plants, in particular of crop plants, such as e.g. of rapeseed, for example, by bathing wounded leaves or pieces of leaf in an agrobacteria solution and then cultivating them in suitable media.
  • the transformation of plants by agrobacteria has been described (White FF (1993) Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu, Academic Press, pp. 15-38 Bens et al. (1993) Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Acadic Press, p.128-143, Potrykus (1991 ) Annu Rev Plant Physiol Plant Molec Biol
  • Stably transformed cells i.e., those containing the introduced DNA integrated into the DNA of the host cell
  • a selectable marker is part of the introduced DNA.
  • any gene capable of conferring resistance to a biocide eg, an antibiotic or herbicide, etc.
  • Transformed cells expressing such a marker gene are capable of being expressed in the presence of concentrations of a marker gene
  • the selection marker allows the selection of transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84)
  • the resulting plants can be grown and crossed in the usual way Two or more generations should be cultured to ensure that genomic integration is stable and hereditary.
  • a complete plant can be obtained using methods known to those skilled in the art. This is exemplified by callus cultures, single cells (eg protoplasts) or leaf discs (Vasil et al., (1984) Cell Culture and Somatic Cell Genetics of Plants, Vol I, Il and IM, Laboratory Procedures and their Applications, Academic Press, Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press). From these still undifferentiated callus cell masses, the formation of shoot and root can be induced in a known manner. The obtained sprouts can be planted out and bred. Corresponding methods have been described (Fennell et al., (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14: 273-278; Jahne et al. (1994) Theor Appl Genet 89: 525-533).
  • the effectiveness of the expression of the transgenically expressed nucleic acids can be determined, for example, in vitro by shoot meristem propagation using one of the selection methods described above.
  • an expression of a target gene which has changed in terms of type and amount and the effect on the phenotype of the plant can be tested on test plants in greenhouse experiments.
  • Another object of the invention relates to transgenic organisms, transformed with at least one inventive expression cassette or a vector of the invention, as well as cells, cell cultures, tissues, parts - such as in plant organisms leaves, roots, etc. - or reproductive derived from such organisms.
  • Organism, starting or host organisms are prokaryotic or eukaryotic organisms, such as, for example, microorganisms or plant organisms. understood. Preferred microorganisms are bacteria, yeasts, algae or fungi.
  • Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus or Cyanobacteria, for example of the genus Synechocystis and others described in pages B-8, A-9, A10 and A11 in Brock Biology of Microorganisms bacterial genera.
  • microorganisms which are capable of infecting plants and thus of transmitting the constructs according to the invention.
  • Preferred microorganisms are those of the genus Agrobacterium and in particular of the species Agrobacterium turnefaciens.
  • Particularly preferred microorganisms are those used for the production of toxins (eg botulinum toxin), pigments (eg carotenoids or flavonoids), antibiotics (eg penicillin), phenylpropanoids (eg tocopherol), polyunsaturated fatty acids (eg arachidonic acid) or vitamins (eg vitamin B12) are capable.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula, Phaffia rhodozyma or Pichia.
  • Preferred fungi are Aspergillus, Trichoderma, Blakeslea, Ashbya, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B. VoI 37, No 1, 2 (1995) on page 15, Table 6 described mushrooms.
  • Preferred transgenic organisms host or initial organisms are mainly plant organisms.
  • Plant organism or cells derived from it generally means any cell, tissue, part or propagation material (such as seeds or fruits) of an organism capable of photosynthesis. Included within the scope of the invention are all genera and species of higher and lower plants of the plant kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred.
  • Plant in the context of the invention means all genera and species of higher and lower plants of the plant kingdom. Included within the term are the mature plants, seeds, shoots and seedlings, as well as derived parts, propagation material (for example tubers, seeds or fruits), plant organs, tissues, protoplasts, callus and other cultures, for example cell or callus cultures, as well all other types of groupings of plant cells into functional or structural units. Mature plants means plants to any developmental Stage beyond the seedling. Keimling means a young, immature plant at an early stage of development.
  • Vegetable organisms according to the invention are also further photosynthetic active organisms, such as algae, cyanobacteria and mosses.
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricomatum, Pirellula, Volvox or Dunaliella.
  • plant organisms are preferably selected from the group of flowering plants (Phylum Anthophyta "angiosperms"). Includes all annual and perennial, monocotyledonous and dicotyledonous plants.
  • the plant is preferably selected from the following plant families: Amaranthaceae, Amaryllidaceae, Asteraceae, Berberidaceae, Brassicaceae, Cannabaceae, Caprifoliaceae, Cargophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Fabaceae, Gentianaceae, Geraniaceae, Illiaceae, Labiatae, Lamiaceae, Leguminosae, Liliaceae , Linaceae, Papaveraceae, Papilloideae, Liliaceae, Linaceae, Malvaceae, Oleaceae, Orchidaceae, Poaceae, Primulaceae, Ranunculaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniacea, Theaceae, Tropae
  • the invention is most preferably applied to dicotyledonous plant organisms.
  • Preferred dicotyledonous plants are especially selected from the dicotyledonous crops, such as the following
  • Rosaceae (roses, apples, almonds, strawberries)
  • Asteraceae especially the genus Lactuca, especially the species sativa (lettuce), as well as sunflower, dandelion, Tagetes or Calendula and others more,
  • Cruciferae Brassicaceae
  • Brassicaceae especially the genus Brassica, especially the species napus (rapeseed), campestris (turnip), oleracea (eg cabbage, cauliflower or broccoli and other cabbages); and the genus Arabidopsis, especially the species thaliana, as well as watercress, radish, canola and others,
  • Cucurbitaceae such as melon, pumpkin, cucumber or zucchini and more
  • Leguminosae especially the genus Glycine, especially the species max (soybean) soy as well as alfalfa, pea, bean plants, lupine or peanut and others more,
  • Rubiaceae preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee shrub) and others more
  • Solanaceae in particular the genus Lycopersicon, more particularly the species esculentum (tomato) and the genus Solanum, more particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, in particular the annum species (paprika), and tobacco , Petunia and more,
  • Sterculiaceae preferably of the subclass Dilleniidae such as, for example, The obroma cacao (cocoa bush) and others more
  • Theaceae preferably of the subclass Dilleniidae such as Camellia sinensis or Thea sinensis (tea shrubs) and others more
  • Umbelliferae (Apiaceae), especially the genus Daucus (especially the species carota), Apium (especially the species graveolens dulce (celery)) as well as parsley and others;
  • monocot plants are also suitable.
  • these are selected from the monocotyledonous crops, such as the families
  • Bromeliaceae pineapple, Spanish moss
  • Cyperaceae sedges
  • - Liliaceae lillies, tulips, hyacinths, onion, garlic
  • Orchidaceae orchids
  • Poaceae grasses, bamboos, corn, sugarcane, wheat
  • Iridaceae irises, gladioli, crocuses
  • gramineae such as rice, maize, wheat or other cereals such as barley, millet, rye, triticale or oats as well as sugar cane and all kinds of grasses.
  • Very particularly preferred plants are selected from the group of plant species Mangold, Tagetes errecta, Tagetes patula, Acacia, Aconitum, Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia, Calendula, Caltha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum , Citrus, Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus, Dimorphotheca, Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania, Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea, Helenium, Helianthus, Hepatica , Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kenya, Laburnum, Lathyrus, Leontodon, Liidium
  • Calendula Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium, Tropaeolum or Adonis.
  • the expression of a particular nucleic acid by a promoter with specificity for the organs of the flower can lead to the formation of sense RNA, antisense RNA or double-stranded RNA in the form of an inverse repetition (dsRNAi).
  • the sense RNA can be translated as a result into specific polypeptides.
  • the antisense RNA and dsRNAi the expression of certain genes can be down-regulated.
  • dsRNAi double-stranded RNA
  • the specificity of the expression constructs and vectors according to the invention for plant flowers is particularly advantageous.
  • Flowering has a role in attracting beneficial insects by incorporation of pigment or synthesis of volatile chemicals.
  • the plant's natural defense mechanisms are insufficient, for example, against pathogens.
  • the introduction of foreign genes from plants, animals, or microbial sources can strengthen the defense. Examples are the protection against insect feeding in tobacco by expression of the Bacillus thuringiensis endotoxin (Vaeck et al. (1987) Nature 328: 33-37) or the protection of the tobacco against fungal attack by expression of a chitinase from the bean (Broglie et al. (1991) Science 254: 1194-1197).
  • a concentrated expression of the corresponding transgene-to-express nucleic acid sequence is advantageous, especially in the outermost envelope of the flower.
  • Constitutive expression in the entire plant may, for example, jeopardize the effect by dilution or impair the growth of the plant or the quality of the plant product.
  • constitutive expression can lead to increased shutting down of the transgene ("gene silencing").
  • promoters with specificity for flowering are advantageous.
  • the person skilled in the art is familiar with a multiplicity of proteins whose recombinant expression in flowering is advantageous.
  • genes are known to those skilled in the art, by the re-priming or elimination by expression of a corresponding antisense RNA also advantageous effects can be achieved. Examples of non-limiting beneficial effects include the achievement of resistance to abiotic stress factors (heat, cold, drought, increased humidity, environmental toxins, UV radiation) and biotic stress factors (pathogens, viruses, insects and diseases) Improving food or feed properties, improving the growth rate or yield, achieving a longer or earlier flowering period, altering or enhancing the fragrance or coloring of the flowers.
  • abiotic stress factors heat, cold, drought, increased humidity, environmental toxins, UV radiation
  • pathogens, viruses, insects and diseases pathogens, viruses, insects and diseases
  • Flavonoid biosynthesis eg chalcone synthases, phenylalanine ammonium lyases
  • DNA repair eg photolyases, Sakamoto A et al. (1998) DNA SEQ 9 (5-6): 335-40
  • isoprenoid biosynthesis eg deoxyxylulose-5-phosphate synthases
  • IPP synthesis eg carotenoid biosynthesis
  • phytoene synthases eg phytoene synthases, phytoene desaturases, lycopene cyclases, hydroxylases or ketolases.
  • Preference is given to nucleic acids which are known for Arabidopsis thaliana chalcone synthase (GenBank Acc.
  • genes of mannitol or trehalose synthesis e.g., trehalose phosphate synthases, trehalose phosphate phosphatases, WO 97/42326); or by inhibition of genes such as trehalase (WO 97/50561).
  • trehalose phosphate synthases e.g., trehalose phosphate synthases, trehalose phosphate phosphatases, WO 97/42326
  • genes such as trehalase WO 97/50561.
  • nucleic acids which are suitable for the transcriptional activator CBFI from Arabidopsis thana-Nana (GenBank Acc. No .: U77378) or the antifreeze protein from Myoxocephalus octodecemspinosus (GenBank Acc. No .: AF306348) or encode functional equivalents thereof.
  • Jasmonic acid or ethylene Jasmonic acid or ethylene, lysozymes from non-plant sources such as T4 lysozyme or lysozyme from various mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, ⁇ -amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), glucanases, lectins (eg phytohemagglutinin, snowdrop lectin, wheat germ agglutinin ), RNAses or ribozymes. Particular preference is given to nucleic acids which are suitable for the chit42 enzymease from Trichoderma harzianum (GenBank Acc.
  • transport proteins that enhance the uptake of metabolites, nutrients or water into the flower and thus optimize flower growth, metabolite composition or yield, for example by expression of an amino acid transporter that accelerates the uptake of amino acids, or a monosaccharide transporter that promotes the uptake of sugars.
  • amino acid transporter that accelerates the uptake of amino acids
  • monosaccharide transporter that promotes the uptake of sugars.
  • nucleic acids which code for the cationic amino acid transporter from Arabidopsis thaliana (GenBank Acc. No .: X92657) or for the monosaccharide transporter from Arabidopsis thaliana (GenBank Acc. No .: AJ002399) or functional equivalents thereof.
  • genes which cause an accumulation of fine chemicals such as tocopherols, tocotrienols, phenylpropanoids, isoprenoids or carotinides, in the flower.
  • examples include the deoxyxylulose-5-phosphate synthases, phytoene synthases, lycopene b-cyclases and the b-carotene tolases.
  • Preference is given to nucleic acids which code for Haematoccus pluvialis NIES-144 (Acc No. D45881) ketolase or functional equivalents thereof.
  • a) carotenoids and / or phenylpropanoids e.g. by optimizing the floral inherent metabolic pathways e.g. by expression of enzymes and regulators of isoprenoid biosynthesis.
  • Preference is given to nucleic acids which are suitable for the Arabidopsis thaliana chalcone synthase (GenBank Acc. No .: M20308), the Arabidopsis thaliana 6-4 photolyase (GenBank Acc.No.:BAB00748) or the blue-light photoreceptor / photolyase homologue (PHHI). from Arabidopsis thaliana (GenBank Acc. No .: U62549) or functional equivalents thereof.
  • nucleic acids which bring about enzymes and regulators of isoprenoid biosynthesis such as deoxyxylulose-5-phosphate synthases and carotenoid biosynthesis, such as the phytoene synthases, lycopene cyclases and ketolases, such as tocopherols, tocotrienols, phenylpropanoids, isoprenoids or carotinides.
  • deoxyxylulose-5-phosphate synthases phytoene synthases, lycopene cyclases and the carotene toetolases.
  • the invention further relates to the use of the transgenic organisms according to the invention described above and the cells, cell cultures, parts derived therefrom - such as, for example, roots, leaves etc. in transgenic plant organisms, and transgenic propagation material such as seeds or fruits, for the production of food or feed, pharmaceuticals or fine chemicals.
  • This process is widely applicable to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavorings, flavorings and dyes.
  • the breeding of the transformed host organisms and the isolation from the host organisms or from the culture medium is carried out by methods known to the person skilled in the art.
  • SEQ ID NO: 1 2554bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g33370
  • SEQ ID NO: 4 2103bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g22430
  • SEQ ID NO: 5 Functionally equivalent fragment (1376 bp) of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus
  • SEQ ID NO: 7 2945 bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At1g26630
  • SEQ ID NO: 9 Functionally equivalent fragment (587 bp) of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus
  • SEQ ID NO: 10 2572bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At4g35100
  • SEQ ID NO: 11 2421 bp fragment of promoter and 5'-untranslated region of the Arabidopsis thaliana gene locus At3g04290
  • SEQ ID NO: 12 2345 bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g46110
  • SEQ ID NO: 13 oligonucleotide primer M1as
  • SEQ ID NO: 14 oligonucleotide primer M1s
  • SEQ ID NO: 15 oligonucleotide primer Miss 16.
  • SEQ ID NO: 16 oligonucleotide primer M1svl
  • SEQ ID NO: 17 oligonucleotide primer M2as
  • SEQ ID NO: 18 oligonucleotide primer M2s
  • SEQ ID NO: 20 oligonucleotide primer M2svl
  • SEQ ID NO: 21 oligonucleotide primer M3as
  • SEQ ID NO: 22 oligonucleotide primer M3s
  • SEQ ID NO: 23 oligonucleotide primer M3ss
  • SEQ ID NO: 24 oligonucleotide primer M3svl
  • SEQ ID NO: 25 oligonucleotide primer M4as
  • SEQ ID NO: 26 oligonucleotide primer M4s
  • SEQ ID NO: 27 oligonucleotide primer M5as
  • SEQ ID NO: 28 oligonucleotide primer M5s
  • SEQ ID NO: 29 oligonucleotide primer M6as
  • SEQ ID NO: 30 oligonucleotide primer M6s
  • oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet & Voet (1995), 2nd edition, Wiley Press New York, pages 896-897).
  • the cloning steps carried out within the scope of the present invention such as, for example, restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of E. coli cells, cultivation of bacteria, propagation of phages and sequence analysis recombinant DNA, as in Sambrook et al. (1989) CoId Spring Harbor Laboratory Press; ISBN 0-87969-309-6 described.
  • the sequencing of recombinant DNA molecules is carried out using a ABI laser fluorescence DNA sequencer according to the method of Sanger (Sanger et al. (1977) Pro Natl Acad. USA 74: 5463-5467).
  • Agrobacterium tumefaciens (strain C58C1 pMP90) is transformed with various promoter-GUS vector constructs. The Agrobacterium strains are then used to produce transgenic plants. For this purpose, a single transformed Agrobacterium colony is incubated overnight in a 4 ml culture (medium: YEB medium with 50 ⁇ g / ml kanamycin and 25 ⁇ g / ml rifampicin) at 28 ° C.
  • This culture is then followed by a 400 ml culture in the same medium seeded, incubated overnight (28 ° C, 220 rpm) and centrifuged (GSA rotor, 8,000 rpm, 20 min) .
  • the pellet is placed in infiltration medium (1/2 MS medium, 0.5 g / l MES, pH 5.8, 50 g / l sucrose), and the suspension is placed in a plant box (Duchefa) and 100 ml of SILVET L-77 (polyalkylene oxide-modified heptamethyltrisiloxane; Osi Specialties Inc., Cat.
  • P030196 The plant box containing 8 to 12 plants is subjected to a vacuum in a desiccator for 10 to 15 minutes followed by spontaneous aeration, this is repeated 2 to 3 times, after which all plants are planted in pots planted with damp soil and under long-day conditions (16 h illumination) (daytime temperature 22 to 24 ° C, night temperature 19 ° C; 65% relative humidity). After 6 weeks, the seeds are harvested.
  • Example 1 Plant growth conditions for tissue-specific RT-PCR analysis
  • 100 seeds are sterilized as described above, incubated at 4 ° C for 4 days and then in 250 ml bottles with MS medium (Sigma M5519) with the addition of another 3% sucrose and 0.5 g / l MES (Sigma M8652), cultivated pH 5.7.
  • the seedlings are grown in a 16-hour light / 8-hour dark cycle (Philips 58W / 33 white light bulbs) at 22 ° C, 120 rpm and harvested after 3 weeks.
  • the seeds are sown on unit soil (VM type, Manna Italia, Via S. Giacomo 42, 39050 San Giacomo / Laives, Bolzano, Italy), incubated for 4 days at 4 ° C.
  • bacterial ⁇ -glucuronidase may be mentioned (Jefferson et al. (1987) EMBO J 6: 3901-3907).
  • the ⁇ -glucuronidase activity can be determined in planta by means of a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid as part of an activity staining (Jefferson et al. (1987) Plant Mol Biol Rep 5: 387-405).
  • a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid as part of an activity staining (Jefferson et al. (1987) Plant Mol Biol Rep 5: 387-405).
  • the plant tissue is cut, embedded, stained, and analyzed as described (e.g., Baumlein et al., (1991) Mol Gen Genet 225: 121-128).
  • the substrate used is MUG (methylumbelliferylglucuronide), which is cleaved into MU (methylumbelliferone) and glucuronic acid. Under alkaline conditions, this cleavage can be monitored quantitatively by fluorometry (excitation at 365 nm, measurement of the emission at 455 nm, SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described (Bustos MM et al. (1989) Plant Gell 1: 839-853). ,
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 is isolated.
  • Arabidopsis thaliana (ecotype Landsberg erecta) native DNA is extracted as described (Galbiati M et al., Funct., Integr. Genomics 2000, 20 1: 25-34).
  • the isolated DNA is used as template DNA in a PCR using the following oligonucleotide primer combinations and annealing temperatures:
  • the amplification is carried out as follows:
  • oligonucleotides were used as primers, which carry at their 5 ⁇ -Termini phosphate residues.
  • the vector pS0301 contains 3 'of the SmaI site the coding sequence of the GUS reporter gene.
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 5 SEQ ID NO: 6
  • SEQ ID NO: 7 SEQ ID NO Figure 8
  • SEQ ID NO: 9 SEQ ID NO: 10
  • SEQ ID NO: 11 and SEQ ID NO: 12 produced gene fusions from the promoter fragments and the ⁇ -glucuronidase (GUS).
  • the expression of the GUS gene can be visualized by means of histochemical staining methods.
  • the "TAIL-PCR” is performed according to an adapted protocol of the method of Liu et al. (1995) Plant J 8 (3): 457-463 and Tsugeki et al. (1996) Plant J 10 (3): 479-489 (see Fig. 9).
  • a first PCR reaction the following master mix (data per reaction mixture) is used
  • the product of the PCR reaction is diluted 1:50 and 1 ⁇ l of each diluted sample is used for a second PCR reaction (secondary PCR).
  • second PCR the following master mix (data per reaction mixture) is used:
  • PCR product of the previous reaction is diluted 1:10 and 1 ⁇ l of each diluted sample is used for a third PCR reaction (tertiary PCR).
  • master mix data per reaction mixture
  • AD 1 5'-NTCGA (G / C) T (A / T) T (G / C) G (A / T) GTT-3 'AD2: 5'-NGTCGA (G. / C) (A / T) GANA (A / T) GAA-3 'AD5: 5' - (A / T) CAGNTG (A / T) TNGTNCTG-3 '
  • the PCR product is monitored by gel electrophoresis, purified and then sequenced as a PCR product.
  • the substrate used is the ⁇ -glucuronidase MUG (methylumbelliferylglucuronide), which is cleaved into MU (methylumbelliferone) and glucuronic acid. Under alkaline conditions, this cleavage can be monitored quantitatively by fluorometry (excitation at 365 nm, measurement of the emission at 455 nm, SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described (Bustos MM et al. (1989) Plant Cell 1: 839-853). ,
  • GUS enzyme activity 25 mg of plant tissue were minced and mixed with extraction buffer (50 mM Na phosphate, pH 7, 10 mM Mecaptoethanol, 10 mM EDTA, 0.1% Triton). The insoluble plant material was sedimented by centrifugation (10000 g, 10 min). 10 ⁇ l each of the supernatant were presented in multi-well plates for the measurement of GUS enzyme activity.
  • extraction buffer 50 mM Na phosphate, pH 7, 10 mM Mecaptoethanol, 10 mM EDTA, 0.1% Triton.
  • reaction buffer extraction buffer + 2 mM methylumbelliferyl- ⁇ -D-glucuronide
  • MU methylumbelliferone

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Abstract

L'invention se rapporte à un procédé d'expression transgénique ciblée de séquences d'acides nucléiques dans les tissus floraux de plantes, ainsi qu'à des cassettes d'expression transgénique et à des vecteurs d'expression qui comportent des promoteurs présentant une spécificité d'expression pour les tissus floraux. Cette invention concerne également des organismes (de préférence des plantes) qui sont transformés au moyen de ces cassettes d'expression transgénique ou vecteurs d'expression, ainsi que des cultures, des parties constitutives, ou des produits de reproduction de ces organismes transformés, et se rapporte en outre à l'utilisation de ces différents éléments pour produire des denrées alimentaires, des produits alimentaires destinés aux animaux, des semences, des substances pharmaceutiques, ou des substances chimiques fines.
EP06754978A 2005-05-04 2006-05-03 Cassettes d'expression transgenique utilisees pour l'expression d'acides nucleiques dans des tissus floraux de plantes Withdrawn EP1880011A2 (fr)

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DE102005021365A DE102005021365A1 (de) 2005-05-04 2005-05-04 Transgene Expressionskassetten zur Expression von Nukleinsäuren in Blütengeweben von Pflanzen
PCT/EP2006/062012 WO2006117381A2 (fr) 2005-05-04 2006-05-03 Cassettes d'expression transgenique utilisees pour l'expression d'acides nucleiques dans des tissus floraux de plantes

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EP2199399A1 (fr) 2008-12-17 2010-06-23 BASF Plant Science GmbH Production de cétocaroténoïdes dans les plantes
ES2899176T3 (es) 2015-11-27 2022-03-10 Kws Saat Se & Co Kgaa Plantas tolerantes a las bajas temperaturas
CN116042694A (zh) * 2022-11-24 2023-05-02 中国科学院南京土壤研究所 禾本科狼尾草属植物非组培遗传转化方法

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US5576198A (en) * 1993-12-14 1996-11-19 Calgene, Inc. Controlled expression of transgenic constructs in plant plastids
EP0913469B1 (fr) * 1996-12-27 2008-07-02 Japan Tobacco Inc. Sequences de promoteurs specifiques pour les organes floraux
US7358418B2 (en) * 1999-07-06 2008-04-15 Senesco Technologies, Inc. Isoforms of eIF-5A: senescence-induced eLF5A; wounding-induced eIF-4A; growth eIF-5A; and DHS
AU2001266251A1 (en) * 2000-06-23 2002-01-02 Syngenta Participations Ag Promoters for regulation of plant gene expression
US8022272B2 (en) * 2001-07-13 2011-09-20 Sungene Gmbh & Co. Kgaa Expression cassettes for transgenic expression of nucleic acids
WO2003006660A1 (fr) * 2001-07-13 2003-01-23 Sungene Gmbh & Co. Kgaa Cassettes d'expression pour l'expression transgenique d'acides nucleiques
CA2496300A1 (fr) * 2002-08-20 2004-04-01 Sungene Gmbh & Co. Kgaa Cassettes d'expression transgenique pour l'expression d'acides nucleiques dans une fleur vegetale
EP1606304A4 (fr) * 2003-03-12 2006-09-27 Evogene Ltd Sequences nucleotidiques regulant une expression genique, constructions et methodes d'utilisation de ces sequences
DE102004007623A1 (de) * 2004-02-17 2005-08-25 Sungene Gmbh & Co. Kgaa Promotoren zur Expression von Genen in Tagetes

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WO2006117381A3 (fr) 2007-04-19
CA2607160A1 (fr) 2006-11-09

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