EP1880011A2 - Transgenic expression cartridges for expressing nucleic acids in the flower tissue of plants - Google Patents
Transgenic expression cartridges for expressing nucleic acids in the flower tissue of plantsInfo
- 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
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- nucleic acid
- promoter
- acid sequence
- sequences
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- 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
Methods for the targeted transgenic expression of nucleic acid sequences in the flower tissues of plants are disclosed, as well as transgenic expression cartridges and expression vectors which contain promoters with expression specificity for flower tissues. Also disclosed are organisms (preferably plants) transformed with these transgenic expression cartridges or expression vectors, cultures, parts or propagation elements derived therefrom, and their use for producing foodstuffs, feedstuffs, seeds, pharmaceuticals or fine chemicals.
Description
Transgene Expressionskassetten zur Expression von Nukleinsäuren in Blütengeweben von PflanzenTransgenic expression cassettes for expression of nucleic acids in flower tissues of plants
Beschreibungdescription
Die Erfindung betrifft Verfahren zur gezielten, transgenen Expression von Nukleinsäu- resequenzen in Blütengeweben von Pflanzen, sowie transgene Expressionskassetten und Expressionsvektoren, die Promotoren mit einer Expressionsspezifität für Gewebe der Blüte enthalten. Die Erfindung betrifft ferner mit diesen transgenen Expressions- kassetten oder Expressionsvektoren transformierte Organismen (bevorzugt Pflanzen), davon abgeleitete Kulturen, Teile oder Vermehrungsgut, sowie die Verwendung derselben zur Herstellung von Nahrungs-, Futtermitteln, Saatgut, Pharmazeutika oder Feinchemikalien.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.
Ziel biotechnologischer Arbeiten an Pflanzen ist die Herstellung von Pflanzen mit vorteilhaften, neuen Eigenschaften zum Beispiel zur Steigerung der landwirtschaftlichen Produktivität, zur Qualitätssteigerung bei Nahrungsmitteln oder zur Produktion bestimmter Chemikalien oder Pharmazeutika (Dunwell JM (2000) J Exp Bot 51 Spec No:487-96). Eine Grundvoraussetzung für die transgene Expression bestimmter Gene ist die Bereitstellung pflanzenspezifischer Promotoren. Promotoren sind wichtigeThe 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
Werkzeuge in der Pflanzenbiotechnologie, um die Expression bestimmter Gene in einer transgenen Pflanze zu steuern und so bestimmte Wesensmerkmals der Pflanze zu erzielen.Tools in plant biotechnology to control the expression of certain genes in a transgenic plant and thus to achieve certain characteristics of the plant.
Verschiedene pflanzliche Promotoren sind bekannt, zum Beispiel konstitutive Promotoren wie der Promotor der Nopalinsynthase aus Agrobakterium, der TR-Doppelpromotor oder der Promotor des 35S-Transkriptes des Blumenkohlmosaikvirus (CaMV) (Odell et al.(1985) Nature 313:810-812). Nachteilig bei diesen Promotoren ist, dass sie in fast allen Geweben der Pflanze konstitutiv aktiv sind. Eine gezielte Expression von Genen in bestimmten Pflanzenteilen oder zu bestimmten Entwicklungszeitpunkten ist mit diesen Promotoren nicht möglich.Various plant promoters are known, for example, 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). A disadvantage of these promoters is that they are constitutively active in almost all tissues of the plant. Targeted expression of genes in certain plant parts or at certain development times is not possible with these promoters.
Beschrieben sind Promotoren mit Spezifitäten für verschiedene pflanzliche Gewebe wie Antheren, Ovarien, Blüten, Blätter, Stengel, Wurzeln, Knollen oder Samen. Die Stringenz der Spezifität, als auch die Expressionsaktivität dieser Promotoren ist sehr unterschiedlich.Described are 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.
Die pflanzliche Blüte dient der geschlechtlichen Fortpflanzung der Samenpflanzen. Pflanzliche Blüten - vor allem die Blütenblätter (Petalen) - akkumulieren häufig große Mengen sekundärer Pflanzenstoffe, wie beispielsweise Terpene, Anthocyane, Carotinoide, Alkaloide und Phenylpropanoide, die der Blüte als Duftstoffe, Abwehrstoffe oder als Farbstoffe dienen. Viele dieser Substanzen sind von ökonomischem Interesse. Zu-
dem ist die Blütenknospe und die Blüte der Pflanze ein empfindliches Organ, besonders gegen Stressfaktoren wie Kälte.The plant bloom serves the sexual reproduction of the seed plants. Herbal flowers - especially the petals - often accumulate large amounts of 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.
Der Arabidopsis thaliana Gen-Locus At5g33370 (abgeleitetes Protein GenBank Acc- No.: NP_198322) kodiert für eine putatives GDSL-motif lipase/hydrolase family Protein.The Arabidopsis thaliana gene locus At5g33370 (derived protein GenBank Acc No .: NP_198322) encodes a putative GDSL motif lipase / hydrolase family protein.
Der Arabidopsis thaliana Gen-Locus At5g22430 (abgeleitetes Protein GenBank Acc- No.: NP_568418) kodiert für ein expressed Protein. Der Arabidopsis thaliana Gen- Locus At1g26630 (abgeleitetes Protein GenBank Acc.-No.: NP_173985) kodiert für einen putativen eukaryotic translation initiation factor 5A / elF-5. Der Arabidopsis thaliana Gen-Locus At4g35100 (abgeleitetes Protein GenBank Acc.-No.: NP_195236) kodiert für ein putatives plasma membrane intrinsic protein (SIMIP). Der Arabidopsis thaliana Gen-Locus At3g04290 (abgeleitetes Protein GenBank Acc.-No.: NP_187079) kodiert für ein putatives GDSL-motif lipase/hydrolase family Protein. Der Arabidopsis thaliana Gen-Locus At5g46110 (abgeleitetes Protein GenBank Acc.-No.: NP_568655) kodiert für einen putativen phosphate/triose-phosphate translocator.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. No .: 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.
Die Funktion, Transkriptions- und die Expressionsmuster dieser Gene sind nicht be- schrieben.The function, transcription and expression patterns of these genes are not described.
Blütenspezifische Promotoren, wie beispielsweise der Phytoensynthase Promotor (WO 92/16635), der Promotor des P-rr Gens (WO 98/22593) oder der Promotor des APE- TALA3 Gens (Hill TA et al. (1998) Development 125:1711-1721) sind bekannt. Diese Promotoren weisen jedoch alle einen oder mehrere Nachteile auf, die eine breite Nutzung beeinträchtigen:Flower-specific promoters, such as the phytoene synthase promoter (WO 92/16635), the promoter of the P-rr gene (WO 98/22593) or the promoter of the APE-TALA3 gene (Hill TA et al. (1998) Development 125: 1711- 1721) are known. However, these promoters all have one or more disadvantages that affect broad usage:
1 ) Sie sind innerhalb der Blüte spezifisch für ein oder mehrere Blütengewebe und gewährleisten nicht die Expression in allen Geweben der Blüte.1) They are specific to one or more flower tissues within the flower and do not guarantee expression in all tissues of the flower.
2) Sie sind - wie im Beispiel des an der Blütenentwicklung beteiligten APETALA3 Gens - während der Blütenentwicklung stark reguliert und nicht zu allen Phasen der Blütenentwicklung aktiv.2) They are - as in the example of the APETALA3 gene involved in flower development - heavily regulated during flower development and not active at all stages of flower development.
3) Sie zeigen mitunter starke Nebenaktivitäten in anderen pflanzlichen Geweben.3) They sometimes show strong side activities in other plant tissues.
Es bestand daher die Aufgabe, Verfahren und geeignete Promotoren für die gezielte, transgene Expression von Nukleinsäuren in den Blütengeweben bereitzustellen. Diese Aufgabe wurde insbesondere durch Bereitstellung der Promotoren der Gene mit den Genlokusbezeichnungen At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110 gelöst.
Diese Promotoren zeigen eine Expression in allen Blütenorganen. Dieses Expressionsmuster kann in der Blütenknospe, der Blüte und der seneszenten Blüte beobachtet werden.It was therefore the object to provide methods and suitable promoters for the targeted, transgenic expression of nucleic acids in the flower tissues. This object has been achieved in particular by providing the promoters of the genes with the gene-locality designations At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 and At5g46110. These promoters show expression in all flower organs. This expression pattern can be observed in the flower bud, the flower and the senescent flower.
Ein erster Gegenstand der Erfindung betrifft Verfahren zur gezielten, transgenen Expression von Nukleinsäuresequenzen in Blütengeweben von Pflanzen, wobei nachfolgende Schritte umfasst sind: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:
I. Einbringen einer transgenen Expressionskassette in pflanzliche Zellen, wobei die transgene Expressionskassette mindestens nachfolgende Elemente enthältI. introducing a transgenic expression cassette into plant cells, wherein the transgenic expression cassette contains at least the following elements
a) mindestens eine Promotorsequenz ausgewählt aus der Gruppe von Sequenzen bestehend ausa) at least one promoter sequence selected from the group of sequences consisting of
i) den Promotorsequenzen gemäß SEQ ID NO: 1, SEQ ID NO: 4, SEQi) the promoter sequences according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ
ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
ii) funktionellen Äquivalenten der Promotorsequenzen gemäß SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 und SEQ ID NO: 9 mit im wesentlichen der gleichen Promotoraktivität wie ein Promotor gemäß SEQ ID NO: 1 , SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undii) functional equivalents of the promoter sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9 having substantially the same promoter activity as one A promoter according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
iii) funktionell äquivalenten Fragmenten der Sequenzen unter i) oder ii) mit im wesentlichen der gleichen Promotoraktivität wie eine Promotorsequenz gemäß i) oder ii)iii) functionally equivalent fragments of the sequences under i) or ii) with essentially the same promoter activity as a promoter sequence according to i) or ii)
undand
b) mindestens eine weitere Nukleinsäuresequenz, undb) at least one further nucleic acid sequence, and
c) gegebenenfalls weitere genetische Kontrollelemente,c) where appropriate, other genetic control elements,
wobei mindestens eine Promotorsequenz und eine weitere Nukleinsäuresequenz funktionell miteinander verknüpft sind und die weitere Nukleinsäuresequenz inwherein at least one promoter sequence and a further nucleic acid sequence are functionally linked together and the further nucleic acid sequence in
Bezug auf die Promotorsequenz heterolog ist, undIs heterologous with respect to the promoter sequence, and
II. Auswahl von transgenen Zellen, die besagte Expressionskassette stabil in das Genom integriert enthalten, und
IM. Regeneration von vollständigen Pflanzen aus besagten transgenen Zellen, wobei mindestens eine der weiteren Nukleinsäuresequenz in im wesentlichen allen Blütengeweben exprimiert wird.II. Selection of 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.
Ein weiterer Gegenstand betrifft transgene Expressionskassetten, wie sie z.B. in dem erfindungsgemäßen Verfahren zum Einsatz kommen können. Bevorzugt umfassen die transgenen Expressionskassetten zur gezielten, transgenen Expression von Nuklein- säuresequenzen in Blütengeweben von Pflanzen,Another object of the invention relates to transgenic expression cassettes, such as e.g. can be used in the method according to the invention. Preferably, the transgenic expression cassettes comprise for targeted, transgenic expression of nucleic acid sequences in flower tissues of plants,
a) mindestens eine Promotorsequenz ausgewählt aus der Gruppe von Sequenzen bestehend ausa) at least one promoter sequence selected from the group of sequences consisting of
i) den Promotorsequenzen gemäß SEQ ID NO: 1 , SEQ ID NO: 4, SEQ IDi) the promoter sequences according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID
NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undNO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
ii) funktionellen Äquivalenten der Promotorsequenzen gemäß SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 und SEQ ID NO: 9 mit im wesentlichen der gleichen Promotoraktivität wie ein Promotor gemäß SEQ ID NO: 1 , SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undii) functional equivalents of the promoter sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9 having substantially the same promoter activity as one A promoter according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
iii) funktionell äquivalenten Fragmenten der Sequenzen unter i) oder ii) mit im wesentlichen der gleichen Promotoraktivität wie eine Promotorsequenz gemäß i) oder ii)iii) functionally equivalent fragments of the sequences under i) or ii) with essentially the same promoter activity as a promoter sequence according to i) or ii)
undand
b) mindestens eine weitere Nukleinsäuresequenz, undb) at least one further nucleic acid sequence, and
c) gegebenenfalls weitere genetische Kontrollelemente,c) where appropriate, other genetic control elements,
wobei mindestens eine Promotorsequenz und eine weitere Nukleinsäuresequenz funktionell miteinander verknüpft sind und die weitere Nukleinsäuresequenz in Bezug auf die Promotorsequenz heterolog ist.wherein at least one promoter sequence and another nucleic acid sequence are functionally linked together and the further nucleic acid sequence is heterologous with respect to the promoter sequence.
Die erfindungsgemäßen Expressionskassetten können weitere genetische Kontrollsequenzen und/oder zusätzliche Funktionselemente enthalten.The expression cassettes according to the invention may contain further genetic control sequences and / or additional functional elements.
Bevorzugt können die transgenen Expressionskassetten durch die transgen zu expri- mierende Nukleinsäuresequenz die Expression eines von besagter Nukleinsäuresequenz kodierten Proteins, und/oder die Expression einer von besagter Nukleinsäure-
sequenz kodierten sense-RNA, anti-sense-RNA oder doppelsträngigen RNA ermöglichen.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.
Die erfindungsgemäßen transgenen Expressionskassetten sind besonders vorteilhaft, da sie eine selektive Expression in den Geweben der Blütenknospe und der Blüte der Pflanze gewähren und zahlreiche Anwendungen, wie beispielsweise eine Resistenz gegen Stressfaktoren wie Kälte oder eine gezielte Synthese von sekundäre Pflanzenstoffe ermöglichen. Die Expression ist im wesentlichen konstant über den gesamten Entwicklungszeitraum der Blütenknospe und Blüte.The 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.
Die erfindungsgemäßen transgenen Expressionskassetten, die von ihnen abgeleiteten transgenen Expressionsvektoren und transgenen Organismen können funktionelle Äquivalente zu den unter SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 beschriebenen Promotorsequenzen umfassen.The 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.
Ein weiterer Gegenstand der Erfindung betrifft transgene Expressionsvektoren, die eine der erfindungsgemäßen Expressionskassetten enthalten.Another object of the invention relates to transgenic expression vectors containing one of the expression cassettes of the invention.
Ein weiterer Gegenstand der Erfindung betrifft transgene Organismen, die eine der erfindungsgemäßen Expressionskassetten oder Expressionsvektoren enthalten. Der Organismus kann ausgewählt sein aus der Gruppe bestehend aus Bakterien, Hefen, Pilzen, nicht-menschlichen tierischen und pflanzlichen Organismen oder von diesen abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut, bevorzugt ist der Organismus ausgewählt aus der Gruppe der landwirtschaftlichen Nutz- pflanzen.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.
Ein weiterer Gegenstand der Erfindung betrifft die Verwendung der besagten Organismen oder von diesen abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut zur Herstellung von Nahrungs-, Futtermittel, Saatgut, Pharmazeutika oder Feinchemikalien, wobei die Feinchemikalien bevorzugt Enzyme, Vitamine, Aminosäuren, Zucker, gesättigte oder ungesättigte Fettsäuren, natürliche oder synthetische Geschmacks-, Aroma- oder Farbstoffe sind. Erfindungsgemäß umfasst sind ferner Verfahren zur Herstellung besagter Nahrungs-, Futtermitteln, Saatgut, Pharmazeutika oder Feinchemikalien unter Einsatz der erfindungsgemäßen Organismen oder von diesen abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut.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.
Die Promotoraktivität eines funktionell äquivalenten Promotors wird als "im wesentlichen gleich" bezeichnet, wenn die Transkription einer bestimmten transgen zu expri- mierenden Nukleinsäuresequenz unter Kontrolle des besagten funktionell äquivalenten Promotors unter ansonsten unveränderten Bedingungen eine gezielte Expression in im wesentlichen allen Blütengeweben zeigt.
"Blüte" meint allgemein einen Spross begrenzten Wachstums, dessen Blätter zu Fortpflanzungsorganen umgewandelt sind. Die Blüte besteht aus verschiedenen "Blütengeweben" wie z.B. den Kelchblätter (Sepalen), den Kronblätter (Petalen), den Staubblätter (oder Staub"gefäßen"; Stamina) oder den Fruchtblätter (Karpellen). Als Androe- ceum wird in der Blüte die Gesamtheit der Staubblätter (Stamina) bezeichnet. Die Staubblätter befinden sich innerhalb des Petalen- bzw. Sepalenkreises. Ein Staubblatt gliedert sich in ein Filament und eine am Ende sitzende Anthere. Diese wiederum unterteilt sich in zwei Theken, welche durch ein Konnektiv miteinander verbunden sind. Jede Theke besteht aus je zwei Pollensäcken, in denen der Pollen gebildet wird.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 Sepalenkreises. 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.
"Im wesentlichen alle Blütengewebe" meint in Bezug auf die Blütengewebe, dass einzelne dieser Gewebe insgesamt oder zu bestimmten Zeitpunkten der Entwicklung keine wesentliche Expression aufweisen können, wobei der Anteil dieser Gewebe jedoch bevorzugt weniger als 20 Gew-%, bevorzugt weniger als 10 Gew.-%, besonders be- vorzugt weniger als 5 Gew.-%, ganz besonders bevorzugt weniger als 1 Gew-% an dem Gesamtgewicht der Blütengeweben beträgt."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.
"Gezielt" meint in Bezug auf die Expression in Blütengeweben bevorzugt, dass die Expression unter Kontrolle eines der erfindungsgemäßen Promotoren in den Blütenge- weben bevorzugt mindestens das doppelte, ganz besonders bevorzugt mindestens das zehnfache, am meisten bevorzugt mindestens das hundertfache beträgt als in einem Nicht-Blütengewebe wie beispielsweise den Blättern."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.
Dass die erfindungsgemäßen Promotoren "im wesentlichen keine Expression in den Pollen und Ovarien zeigen", meint bevorzugt, dass die der statistischen Mittelwert der Expression über alle reproduktiven Blütengewebe maximal 10 %, bevorzugt maximal 5 %, am meisten bevorzugt maximal 1% des statistischen Mittelwerts der Expression über alle Blütengewebe unter den gleichen Bedingungen beträgt.The fact that 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.
Bevorzugt ist die Expression innerhalb der Blütengewebe im wesentlichen konstant. "Im wesentlichen Konstant" bedeutet dabei bevorzugt, dass die Standartabweichung der Expression zwischen den einzelnen Blütengeweben bezogen auf den statistischen Mittelwert der Expression über alle Blütengewebe geringer ist als 50 %, bevorzugt 20 %, besonders bevorzugt 10 %, ganz besonders bevorzugt 5 %.Preferably, 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%.
Bevorzugt ist die Expression innerhalb mindestens eines bestimmten Blütengewebes über alle Entwicklungsstufen der Blüte im wesentlichen konstant. "Im wesentlichen konstant" bedeutet dabei bevorzugt, dass die Standartabweichung der Expression zwischen den einzelnen Entwicklungszeitpunkten des jeweiligen Blütengewebes bezogen auf den statistischen Mittelwert der Expression über alle Entwicklungszeitpunkte geringer ist als 50 %, bevorzugt 20 %, besonders bevorzugt 10 %, ganz besonders bevorzugt 5 %.
Bevorzugt werden im Rahmen der Ermittlung der Expressionshöhe solche Nukleinsäu- resequenzen in funktioneller Verknüpfung mit dem zu prüfenden Promotor eingesetzt, die für leicht quantifizierbare Proteine kodieren. Ganz besonders bevorzugt sind dabei Reporterproteine (Schenborn E, Groskreutz D (1999) Mol Biotechnol 13(1): 29-44) wie "green fluorescence protein" (GFP) (Chui WL et al. (1996) Curr Biol 6:325-330; Leffel SM et al.(1997) Biotechniques 23(5):912-8), Chloramphenicoltransferase, Luziferase (Miliar et al. (1992) Plant Mol Biol Rep 10:324-414), ß-Glucuronidase oder b-Galactosi- dase. Ganz besonders bevorzugt ist die ß-Glucuronidase (Jefferson et al. (1987) EM- BO J 6:3901-3907).Preferably, 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 %. Within the scope of the determination of the expression level, preference is given to using those nucleic acid sequences in functional linkage with the promoter to be tested which code for readily quantifiable proteins. Very particular preference is given to reporter proteins (Schenborn E, Groskreutz D (1999) Mol Biotechnol 13 (1): 29-44) such as "green fluorescence protein" (GFP) (Chui WL et al. (1996) Curr Biol 6: 325- 330; Leffel SM et al. (1997) Biotechniques 23 (5): 912-8), chloramphenicol transferase, luciferase (Miliar et al., (1992) Plant Mol Biol Rep 10: 324-414), β-glucuronidase or β-galactosi - that. Very particular preference is given to β-glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
"Ansonsten unveränderte Bedingungen" bedeutet, dass die Expression, die durch eine der zu vergleichenden transgenen Expressionskassetten initiiert wird, nicht durch Kombination mit zusätzlichen genetischen Kontrollsequenzen, zum Beispiel Enhancer- Sequenzen, modifiziert wird. Unveränderte Bedingungen bedeutet ferner, dass alle Rahmenbedingungen wie beispielsweise Pflanzenart, Entwicklungsstadium der Pflanzen, Zuchtbedingungen, Assaybedingungen (wie Puffer, Temperatur, Substrate etc.) zwischen den zu vergleichenden Expressionen identisch gehalten werden."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.
"Transgen" meint - zum Beispiel bezüglich einer Expressionskassette, oder einen diese umfassenden Expressionsvektor oder transgenen Organismus, alle solche durch gentechnische Methoden zustandegekommene Konstruktionen, in denen sich entwederBy "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
a) der Promotor gemäß SEQ ID NO: 1, 4, 7, 10, 11 und 12 oder ein funktionelles Äquivalent desselben oder ein Teil der vorgenannten, odera) the promoter according to SEQ ID NO: 1, 4, 7, 10, 11 and 12 or a functional equivalent thereof or a part of the aforementioned, or
b) eine mit a) funktionell verknüpfte weitere Nukleinsäuresequenz, oderb) a further nucleic acid sequence functionally linked to a), or
c) (a) und (b)c) (a) and (b)
sich nicht in ihrer natürlichen, genetischen Umgebung befinden oder durch gentechnische Methoden modifiziert wurden, wobei die Modifikation beispielhaft eine Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer Nukleoti- dreste sein kann. Bevorzugt ist die in den Expressionskassetten enthaltene erfin- dungsgemäße Promotorsequenz (z.B. die Sequenz gemäß SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 oder 12) heterolog in Bezug auf die mit ihr funktionell verknüpfte, trans- gen zu exprimierende weitere Nukleinsäuresequzenz. "Heterolog" meint in diesem Zusammenhang, dass die weitere Nukleinsäuresequenz nicht für das Gen kodiert, das natürlicherweise unter der Kontrolle des besagten Promotors steht.are not in their natural genetic environment or have been modified by genetic engineering methods, which modification may exemplarily be substitutions, additions, deletions, inversions or insertions of one or more nucleotides. The promoter sequence according to the invention contained in the expression cassettes (for example the sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) is preferably heterologous with respect to FIG their functionally linked, transgene-expressing further Nukleinsäuresequzenz. "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.
"Natürliche genetische Umgebung" meint den natürlichen chromosomalen Locus in dem Herkunftsorganismus oder das Vorliegen in einer genomischen Bibliothek. Im Fall
einer genomischen Bibliothek ist die natürliche, genetische Umgebung der Nukleinsäu- resequenz bevorzugt zumindest noch teilweise erhalten. Die Umgebung flankiert die Nukleinsäuresequenz zumindest an einer Seite und hat eine Sequenzlänge von mindestens 50 bp, bevorzugt mindestens 500 bp, besonders bevorzugt mindestens 1000 bp, ganz besonders bevorzugt mindestens 5000 bp. Eine natürlich vorkommendes Expressionskassette - beispielsweise die natürlich vorkommende Kombination des Promotors eines Gens kodierend für ein Protein gemäß der Gene mit den Genlokusbezeichnungen At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110 oder ein funktionelles Äquivalent desselben mit seinen entsprechen- den kodierenden Sequenzen wird zu einem transgenen Expressionskonstrukt, wenn diese durch nicht-natürliche, synthetische ("künstliche") Verfahren wie beispielsweise einer Mutagenisierung geändert wird. Entsprechende Verfahren sind beschrieben (US 5,565,350; WO 00/15815; siehe auch oben)."Natural genetic environment" means the natural chromosomal locus in the lineage or the presence in a genomic library. In the case 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. Corresponding methods are described (US 5,565,350, WO 00/15815, see also above).
"Transgen" meint in Bezug auf eine Expression ("transgene Expression") bevorzugt all solche unter Einsatz einer transgenen Expressionskassette, eines transgenen Expressionsvektors oder transgenen Organismus - entsprechend dem oben gegebenen Definitionen - realisierten Expressionen."Transgene" in terms of expression ("transgenic 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.
"Funktionelle Äquivalente" eines Promotors gemäß SEQ ID NO: 1 , 4, 7, 10,11 und 12 meint insbesondere natürliche oder künstliche Mutationen eines Promotors, beispielsweise gemäß SEQ ID NO: 2, 3, 5, 6, 8, und 9, sowie homologe Sequenzen aus anderen Organismen, bevorzugt aus pflanzlichen Organismen, die im wesentlichen die gleiche Promotoraktivität wie einer der Promotoren gemäß SEQ ID NO: 1 , 4, 7, 10,11 oder 12 aufweisen."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.
Funktionelle Äquivalente umfassen auch all die Sequenzen, die von dem komplementären Gegenstrang der durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 definierten Sequenzen abgeleitet sind und im wesentlichen die gleiche Promotoraktivität aufweisen.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.
Funktionelle Äquivalente zu den Promotoren gemäß 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 umfassen bevorzugt solche Sequenzen, dieFunctional equivalents to the promoters according to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 preferably include those sequences which
a) im wesentlichen die gleiche Promotoraktivität wie einer der Promotoren gemäß SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 aufweisen unda) 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
b) die eine Homologie aufweisen von mindestens 50 %, bevorzugt 70 %, vorzugsweise mindestens 80 %, besonders bevorzugt mindestens 90 %, ganz besonders bevorzugt mindestens 95 %, am meisten bevorzugt 99% zu der Sequenz eines der Promotoren gemäß SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12, wobei sich die Homologie über eine Länge von von mindestens 100 Basenpaaren, be-
vorzugt mindestens 200 Basenpaaren, besonders bevorzugt von mindestens 300 Basenpaaren, ganz besonders bevorzugt von mindestens 400 Basenpaaren, am meistens bevorzugt von mindestens 500 Basenpaaren erstreckt.b) which have a homology of at least 50%, preferably 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, most preferably 99% to the sequence of one of the promoters according to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively, wherein the homology is over a length of at least 100 base pairs, preferably at least 200 base pairs, more preferably of at least 300 base pairs, most preferably of at least 400 base pairs, most preferably extending from at least 500 base pairs.
Dabei kann die Expressionshöhe der funktionellen Äquivalente sowohl nach unten als auch nach oben im Vergleich zu einem Vergleichswert abweichen. Bevorzugt sind dabei solche Sequenzen, deren Expressionshöhe, gemessen anhand der transkribierten mRNA oder dem infolge translatierten Protein, unter ansonsten unveränderten Bedingungen quantitativ um nicht mehr als 50 %, bevorzugt 25 %, besonders bevorzugt 10 % von einem Vergleichswert erhalten mit denen durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 beschriebenen Promotoren unterscheidet. Besonders bevorzugt sind solche Sequenzen, deren Expressionshöhe, gemessen anhand der transkribierten mRNA oder dem infolge translatierten Protein, unter ansonsten unveränderten Bedingungen quantitativ um mehr als 50 %, bevorzugt 100 %, besonders bevorzugt 500 %, ganz besonders bevorzugt 1000 % einen Vergleichswert erhalten mit dem durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 beschriebenen Promotor übersteigt.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. Particularly preferred are 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 more than 50%, preferably 100%, more preferably 500%, most preferably 1000% obtained a comparison value with the by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 described promoter.
Beispiele für die in den erfindungsgemäßen transgenen Expressionskassetten oder transgenen Expressionsvektoren zum Einsatz kommenden Promotorsequenzen lassen sich beispielsweise in weiteren Organismen, deren genomische Sequenz bekannt ist, wie beispielsweise Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthium annuus, Linum sativum durch Homologievergleiche in Datenbanken leicht auffinden. Bevorzugt kann man dazu von den kodierenden Regionen der Gene ausgehen, deren Promotoren beispielsweise durch SEQ ID NO 1, 4, 7, 10, 11 oder 12 beschrieben sind. Ausgehend von beispielsweise den cDNA Sequenzen die Sequenzen der Gene mit den Genlokusbezeichnungen At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110 können die entsprechenden homologen Gene in anderen Pflanzenarten durch Durchmusterung von Datenbanken oder Genbanken (unter Verwendung von entsprechenden Gensonden) leicht in der dem Fachmann geläufigen Weise identifiziert werden.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. Starting from, for example, the cDNA sequences, 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.
In einer bevorzugten Ausführungsform der Erfindung umfassen funktionelle Äquivalente der Promotoren, beschrieben durch SEQ ID NO: 1, 4, 7, 10, 11 und 12, all solche Promotoren, die sich in einem pflanzlichen Organismus in 5'- Richtung vor einer geno- mischen Sequenz befinden, die für ein Protein mit einer Homologie von mindestens 60%, bevorzugt mindestens 80%, besonders bevorzugt mindestens 90%, am meisten bevorzugt mindestens 95 % kodieren. Bevorzugt handelt es dabei um die Gene mit der Genlokusbezeichnungen At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110 entsprechend den Proteinen mit den Sequenzen gemäß den Acc. No. NPJ98322, NP_568418, NPJ73985, NPJ95236, NPJ87079, NP_568655, wobei besagte Promotoren den natürlichen Promotor der besagten genomischen Sequenz darstellen.
Dem Fachmann sind verschiedene Verfahren bekannt, um ausgehend von einer Nu- kleinsäuresequenz (z.B. einem Gentranskript wie beispielsweise einer cDNA) den Promotor des entsprechenden Genes zu identifizieren und zu isolieren. Dazu stehen bei- spielsweise prinzipiell alle Methoden zur Amplifikation flankierender chromosomaler Sequenzen zur Verfügung. Die beiden am häufigsten genutzten Verfahren sind die inverse PCR ("iPCR"; schematisch dargestellt in Fig. 10) und die "Thermal Asymmetrie Interlaced PCR" ("TAIL PCR").In a preferred embodiment of the invention, 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%. Preferably, 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. NPJ98322, NP_568418, NPJ73985, NPJ95236, NPJ87079, NP_568655, said promoters being the natural promoter of said genomic sequence. The skilled worker is aware of various methods for identifying and isolating the promoter of the corresponding gene starting from a nucleic acid sequence (for example a gene transcript such as a cDNA). In principle, for example, 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").
Für die "iPCR" wird genomische DNA des Organismus, aus dem der funktionell äquivalente Promotor zu isolieren ist, mit einem gegebenen Restriktionsenzym komplett verdaut und anschließend werden in einem verdünnten Ansatz die einzelnen Fragmente rückligiert, also mit sich selbst zu einem ringförmigen Molekül verbunden. In der Vielzahl enstehender ringförmiger DNA-Moleküle befinden sich auch solche, die die be- kannte Sequenz (beispielsweise die Sequenz kodierend für das homologe Protein) enthalten. Ausgehend davon kann das ringförmige Molekül mittels PCR amplifiziert werden, indem ein Primerpaar verwendet wird, bei dem beide Primer sich an den bekannten Sequenzabschnitt anlagern können. Eine Ausführungsmöglichkeit für die „iPCR" ist beispielhaft in Beispiel 4 wiedergegeben.For the "iPCR", 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.
Die "TAIL-PCR" beruht auf der Verwendung von einerseits einem Satz sukzessive verkürzter hochspezifischer Primer, die sich an die bekannte genomische Sequenz (beispielsweise die Sequenz kodierend für das homologe Protein) anlagern, und andererseits einem Satz kürzerer Zufallsprimer mit geringer Schmelztemperatur, so dass eine sequenzunspezifischere Anlagerung an die bekannte genomische Sequenz flankierende genomische DNA erfolgt. Die Anlagerung der Primer an die zu amplifizierende DNA kann mit einer solchen Primerkombimation so gestaltet werden, dass eine spezifische Amplifikation der gewünschten Zielsequenz möglich wird. Eine Ausführungsmöglichkeit für die "TAIL-PCR" ist beispielhaft in Beispiel 4 wiedergegeben.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.
Ein weiterer Gegenstand der Erfindung betrifft Verfahren zur Herstellung einer trans- genen Expressionskassette mit Spezifität für Blütengewebe, umfassend nachfolgende Schritte: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:
I. Isolation eines Promotors mit Spezifität für Blütengewebe, wobei bei der Isolation mindestens eine Nukleinsäuresequenz oder ein Teil derselben zum Einsatz kommt, wobei besagte Nukleinsäuresequenz für eine Aminosäuresequenz kodiert, die mindestens einen Teil der Sequenzen gemäß den Acc. No. NP_198322, NP_568418, NP_173985, NP_195236, NP_187079 oder NP_568655 umfasst;
II. funktionelle Verknüpfung besagten Promotors mit einer weiteren Nukleinsäurese- quenz, wobei besagte Nukleinsäuresequenz in Bezug auf den Promotor heterolog ist.I. 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; II. Functional linkage of said promoter with a further nucleic acid sequence, wherein said nucleic acid sequence is heterologous with respect to the promoter.
Bevorzugt kodiert besagte Nukleinsäuresequenz für eine Aminosäuresequenz umfassend Sequenzen gemäß den Acc. No. NP_198322, NP_568418, NP_173985, NP_195236, NP_187079 oder NP_568655.Preferably, 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.
"Teil" meint in Bezug auf die Nukleinsäuresequenz bevorzugt eine Sequenz von min- destens 10 Basen, bevorzugt 15 Basen, besonders bevorzugt 20 Basen, am meisten bevorzugt 30 Basen. In einer Bevorzugten Ausführungsform basiert das erfindungsgemäße Verfahren auf der Polymerasekettenreaktion, wobei die besagte Nukleinsäuresequenz oder ein Teil derselben als Primer eingesetzt wird. Im Rahmen der funktionellen Verknüpfung können dem Fachmann bekannte Verfahren wie z.B. Ligation etc. eingesetzt werden (s.u.)."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. In a preferred embodiment, 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. In the context of functional linking, methods known to those skilled in the art, such as e.g. Ligation, etc. are used (s.u.).
"Mutation" meint Substitution, Addition, Deletion, Inversion oder Insertion eines oder mehrerer Nukleotidreste. Somit werden beispielsweise auch solche Nukleotidsequen- zen durch die vorliegende Erfindung mit umfasst, welche man durch Modifikation der Promotoren gemäß SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 erhält. Ziel einer solchen Modifikation kann die weitere Eingrenzung der darin enthaltenen Sequenz oder z.B. auch die Einfügung weiterer Restriktionsenzymschnittstellen, die Entfernung überflüssiger DNA oder das Hinzufügen weiterer Sequenzen, zum Beispiel weiterer regulatorischer Sequenzen, sein."Mutation" means substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Thus, for example, 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.
Wo Insertionen, Deletionen oder Substitutionen, wie z.B. Transitionen und Transversionen, in Frage kommen, können an sich bekannte Techniken, wie in vitro- Mutagenese, "primer repair", Restriktion oder Ligation verwendet werden. Transition meint einen Basen paaraustausch eines Purin/Pyrimidin-Paares in ein anderes Pu- rin/Pyrimidin-Paar (z.B. A-T gegen G-C). Transversion meint einen Basenpaaraus- tausch eines Purin/Pyrimidin-Paares gegen ein Pyrimidin/Purin-Paar (z.B. A-T gegen T-A). Deletion meint die Entfernung eines oder mehrerer Basenpaare. Insertion meint die Einführung eines oder mehrerer Basenpaare.Where insertions, deletions or substitutions, e.g. 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.
Durch Manipulationen, wie z.B. Restriktion, "chewing-back" oder Auffüllen von Überhängen für "blunt ends" können komplementäre Enden der Fragmente für die Ligation zur Verfügung gestellt werden. Zu analogen Ergebnissen kann man auch unter Verwendung der Polymerasekettenreaktion (PCR) unter Verwendung spezifischer Oligo- nukleotid-Primer kommen.By manipulations, such as Restriction, chewing-back, or padding for blunt ends may be provided with complementary ends of the fragments for ligation. Analogous results can also be obtained using polymerase chain reaction (PCR) using specific oligonucleotide primers.
Unter Identität zwischen zwei Nukleinsäuren wird die Identität der Nukleotide über die jeweils gesamte Nukleinsäurelänge verstanden, insbesondere die Identität die durch
Vergleich mit Hilfe der Vector NTI Suite 7.1 Software der Firma Informax (USA) unter Anwendung der Clustal Methode (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr;5(2):151-1) unter Einstellung folgender Parameter berechnet wird:By 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:
Multiple alignment parameter:Multiple alignment parameters:
Gap opening penalty 10Gap opening penalty 10
Gap extension penalty 10 Gap Separation penalty ränge 8Gap extension penalty 10 Gap Separation penalty ranks 8
Gap Separation penalty offGap separation penalty off
% identity for alignment delay 40% identity for alignment delay 40
Residue specific gaps offResidue specific gaps off
Hydrophil ic residue gap off Transition weighing 0Hydrophilic deposit gap off Transition weighing 0
Pairwise alignment parameter:Pairwise alignment parameter:
FAST algorithm onFAST algorithm on
K-tuple size 1K-tuple size 1
Gap penalty 3Gap penalty 3
Window size 5Window size 5
Number of best diagonals 5Number of best diagonals 5
Beispielhaft wird unter einer Sequenz, die eine Homologie von mindestens 50 % auf Nukleinsäurebasis z. B. mit der Sequenz SEQ ID NO: 1 aufweist, eine Sequenz verstanden, die bei einem Vergleich mit der Sequenz SEQ ID NO: 1 nach obigem Programmalgorithmus mit obigem Parametersatz eine Homologie von mindestens 50 % aufweist.By way of example, a sequence which has a homology of at least 50% based on nucleic acid z. B. with the sequence SEQ ID NO: 1, understood a sequence having a homology of at least 50% in a comparison with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set.
Unter Identität zwischen zwei Proteinen wird die Identität der Aminosäuren über die jeweils gesamte Proteinlänge verstanden, insbesondere die Identität die durch Vergleich mit Hilfe der Vector NTI Suite 7.1 Software der Firma Informax (USA) unter Anwendung der Clustal Methode (Higgins DG, Sharp PM. Fast and sensitive multiple se- quence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr;5(2):151-1) unter Einstellung folgender Parameter berechnet wird: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:
Multiple alignment parameter:Multiple alignment parameters:
Gap opening penalty 10Gap opening penalty 10
Gap extension penalty 10Gap extension penalty 10
Gap Separation penalty ränge 8
Gap Separation penalty offGap Separation penalty ranks 8 Gap separation penalty off
% identity for alignment delay 40 Residue specific gaps off% identity for alignment delay 40 Residue specific gaps off
Hydrophil ic residue gap offHydrophilic coagulation gap off
Transition weighing 0Transition weighing 0
Pairwise alignment parameter:Pairwise alignment parameter:
FAST algorithm onFAST algorithm on
K-tuple size 1K-tuple size 1
Gap penalty 3Gap penalty 3
Window size 5Window size 5
Number of best diagonals 5Number of best diagonals 5
Unter Homologie zwischen zwei Polypeptiden wird die Identität der Aminosäuresequenz über die jeweilige Sequenzlänge verstanden, die durch Vergleich mit Hilfe des Programmalgorithmus GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) unter Einstellung folgender Parameter berechnet wird:Homology between two polypeptides is understood to mean the identity of the amino acid sequence over the respective sequence length, which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters becomes:
Gap Weight: 8 Length Weight: 2Gap Weight: 8 Length Weight: 2
Average Match: 2,912 Average Mismatch:-2,003Average Match: 2,912 Average Mismatch: -2,003
Beispielhaft wird unter einer Sequenz, die eine Homologie von mindestens 60 % auf Proteinbasis mit den Sequenzen gemäß NP_198322, NP_568418, NP_173985, NPJI95236, NP_187079, NP_568655 aufweisen, eine Sequenz verstanden, die bei einem Vergleich nach obigem Programmalgorithmus mit obigem Parametersatz eine Homologie von mindestens 60 % aufweist.By way of example, 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%.
Funktionelle Äquivalente meint ferner DNA Sequenzen, die unter Standardbedingungen mit einer der Nukleinsäuresequenzen kodierend für einen der Promotoren gemäß SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 oder der zu diesen komplementären Nukleinsäuresequenzen hybridisieren und die im wesentlichen gleichen Promotorei- genschaften haben.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.
Der Begriff der Standardhybridisierungsbedingungen ist breit zu verstehen und meint sowohl stringente als auch weniger stringente Hybridisierungsbedingungen. Solche Hybridisierungsbedingungen sind unter anderem bei Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning - A Laboratory Manual, 2. Auflage, CoId Spring Harbor Laboratory Press, 1989, Seiten 9.31-9.57 oder in Current Protocols in Molecular Biolo- gy, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. beschrieben. Beispielhaft können die
Bedingungen während des Waschschrittes ausgewählt sein aus dem Bereich von Bedingungen begrenzt von solchen mit geringer Stringenz (mit ungefähr 2X SSC bei 50°C) und solchen mit hoher Stringenz (mit ungefähr 0.2X SSC bei 50°C bevorzugt bei 65°C) (2OX SSC: 0,3 M Natriumeitrat, 3 M NaCI, pH 7.0). Darüber hinaus kann die Temperatur während des Waschschrittes von niedrig stringenten Bedingungen bei Raumtemperatur, ungefähr 22°C, bis zu stärker stringenten Bedingungen bei ungefähr 65°C angehoben werden. Beide Parameter, Salzkonzentration und Temperatur, können gleichzeitig variiert werden, auch kann einer der beiden Parameter konstant gehalten und nur der andere variiert werden. Während der Hybridisierung können auch de- naturierende Agenzien wie zum Beispiel Formamid oder SDS eingesetzt werden. In Gegenwart von 50 % Formamid wird die Hybridisierung bevorzugt bei 42°C ausgeführt. Einige beispielhafte Bedingungen für Hybridisierung und Waschschritt sind infolge gegeben:The term 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. By way of example, 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). In addition, 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. During hybridization it is also possible to use denicating agents such as, for example, formamide or SDS. In the presence of 50% formamide, hybridization is preferably carried out at 42 ° C. Some exemplary conditions for hybridization and washing step are given as follows:
(1) Hybridisierungsbedingungen mit zum Beispiel(1) hybridization conditions with, for example
a) 4X SSC bei 65°C, oder b) 6X SSC, 0,5% SDS, 100 μg/ml denaturierte, fragmentierte Lachssperma- DNA bei 65°C, oder c) 4X SSC, 50% Formamid, bei 42°C, oder d) 2X oder 4X SSC bei 50°C (schwach stringente Bedingung), oder e) 2X oder 4X SSC, 30 bis 40% Formamid bei 42°C (schwach stringente Bedingung), oder f) 6x SSC bei 45°C, oder, g) 0,05 M Natriumphosphatpuffer pH 7,0, 2 rtiM EDTA, 1% BSA und 7% SDS.a) 4X SSC at 65 ° C, or b) 6X SSC, 0.5% SDS, 100 μg / ml denatured, fragmented salmon sperm DNA at 65 ° C, or c) 4X SSC, 50% formamide, at 42 ° C , or d) 2X or 4X SSC at 50 ° C (weakly stringent condition), or e) 2X or 4X SSC, 30 to 40% formamide at 42 ° C (weakly stringent condition), or f) 6x SSC at 45 ° C , or, g) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and 7% SDS.
(2) Waschschritte mit zum Beispiel(2) washing steps with for example
a) 0,1 X SSC bei 65°C, oder b) 0,1 X SSC, 0,5% SDS bei 68°C, oder c) 0, 1 X SSC, 0,5% SDS, 50% Formamid bei 42°C, oder d) 0,2X SSC, 0, 1 % SDS bei 42°C, oder e) 2X SSC bei 65°C (schwach stringente Bedingung), oder f) 40 rtiM Natriumphosphatpuffer pH 7,0, 1% SDS, 2 rtiM EDTA.a) 0.1 X SSC at 65 ° C, or b) 0.1 X SSC, 0.5% SDS at 68 ° C, or c) 0.1 X SSC, 0.5% SDS, 50% formamide at 42 ° C, or d) 0.2X SSC, 0.1% SDS at 42 ° C, or e) 2X SSC at 65 ° C (weak stringent condition), or f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.
Verfahren zur Herstellung erfindungsgemäßer funktioneller Äquivalente umfassen bevorzugt die Einführung von Mutationen in einen der Promotoren gemäß SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12. Eine Mutagenese kann ungerichtet ("random") erfolgen, wobei die mutagenisierten Sequenzen anschließend bezüglich ihrer Eigen- schatten nach einer "trial-and-error" Prozedur durchmustert werden. Besonders vorteilhafte Selektionskriterien umfassen beispielsweise die Höhe der resultierenden Expression der eingeführten Nukleinsäuresequenz in einem Blütengewebe.
Verfahren zur Mutagenisierung von Nukleinsäuresequenzen sind dem Fachmann bekannt und schließen beispielhaft die Verwendung von Oligonukleotiden mit einer oder mehr Mutationen im Vergleich zu der zu mutierenden Region ein (z.B. im Rahmen ei- ner "site-specific mutagenesis"). Typischerweise kommen Primer mit ungefähr 15 bis ungefähr 75 Nukleotiden oder mehr zum Einsatz, wobei bevorzugt ca. 10 bis ca. 25 oder mehr Nukleotidreste an beiden Seiten der zu verändernden Sequenz lokalisiert sind. Details und Durchführung besagter Mutageneseverfahren sind dem Fachmann geläufig (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). Eine Mutagenese kann auch durch Behandlung von beispielsweise transgenen Expressionsvektoren, die eine der erfindungsgemäßen Nukleinsäuresequenzen enthalten, mit mutagenisierenden Agentien wie Hydroxylamin realisiert werden.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"). Typically, 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.
Alternativ können nicht-essentielle Sequenzen eines erfindungsgemäßen Promotors deletiert werden, ohne die genannten wesentlichen Eigenschaften signifikant zu beeinträchtigen. Derartige Deletionsvarianten stellen funktionelle Äquivalente zu den Promotoren, beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12, oder zu funktionellen Äquivalenten derselben dar. Die Eingrenzung der Promotorsequenz auf bestimmte, essentielle regulatorische Regionen kann z.B. mit Hilfe von Suchroutine zur Suche von Promotorelementen vorgenommen werden. Oft sind in den für die Promotoraktivität relevanten Regionen bestimmte Promotorelemente gehäuft vorhanden. Diese Analyse kann beispielsweise mit Computerprogrammen wie dem Programm PLACE ("Plant Cis-acting Regulatory DNA Elements"; Higo K et al. (1999) Nucl Acids Res 27(1 ): 297-300), der BIOBASE Datenbank "Transfac" (Biologische DatenbankenAlternatively, non-essential sequences of a promoter according to the invention can be deleted without significantly impairing the abovementioned essential properties. Such 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. This analysis can be carried out, for example, with computer programs such as the program PLACE ("Plant Cis-acting Regulatory DNA Elements", Higo K et al., (1999) Nucl Acids Res 27 (1): 297-300), the BIOBASE database "Transfac" (Biological databases
GmbH, Braunschweig; Wingender E et al. (2001) Nucleic Acids Res 29(1):281-3) oder Datenbank PlantCARE (Lescot M et al. (2002) Nucleic Acids Res 30(1):325-7) vorgenommen werden.GmbH, Brunswick; Wingender E et al. (2001) Nucleic Acids Res 29 (1): 281-3) or PlantCARE database (Lescot M et al. (2002) Nucleic Acids Res 30 (1): 325-7).
Bevorzugt umfassen die funktionell äquivalenten Fragmente eines der erfindungsgemäßen Promotoren - beispielsweise der Promotoren beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 - mindestens 200 Basenpaar, ganz besonders bevorzugt mindestens 500 Basenpaare, am meisten bevorzugt mindestens 1000 Basenpaare des 3'-Endes des jeweiligen erfindungsgemäßen Promotors - beispielsweise der Promotoren beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12 -, wobei die Länge vom Transkriptionsstart ("ATG"-Kodon) in 5'-Richtung stromaufwärts gerechnet wird. Ganz besonders bevorzugte funktionelle Äquivalente sind die Promotorsequenzen beschrieben durch SEQ ID NO: 2, 3, 5, 6, 8 oder 9. Weitere funktionell äquivalente Fragmente können beispielsweise durch Deletion eventuell noch vorhan- dener 5'-untranslatierter Bereiche erzeugt werden. Zu diesem Zweck kann derPreferably, 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
Transkriptionsstart der entsprechenden Gene durch dem Fachmann geläufige Verfah-
ren (wie beispielsweise 5'-RACE) bestimmt und die 5'-untranslatierten durch PCR- vermittelte Methoden oder Endonukleaseverdau deletiert werden.Start of transcription of the corresponding genes by methods familiar to the person skilled in the art. ren (such as 5'-RACE) are determined and the 5'-untranslated be deleted by PCR-mediated methods or Endonukleaseverdau.
In erfindungsgemäßen transgenen Expressionskassetten steht mindestens einer der erfindungsgemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12) in funktioneller Verknüpfung mit mindestens einer transgen zu exprimierenden Nukleinsäuresequenz.In transgenic expression cassettes according to the invention, at least 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) is in functional linkage with at least one transgenic to be expressed nucleic acid sequence.
Unter einer funktionellen Verknüpfung versteht man zum Beispiel die sequentielle An- Ordnung eines der erfindungsgemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12) mit einer transgen zu exprimierenden Nukleinsäuresequenz und ggf. weiterer genetischer Kontrollsequenzen wie zum Beispiel einem Terminator oder einer Polyadenylierungssequenz derart, dass der Promotor seine Funktion bei der transgenen Expression der Nukleinsäuresequenz unter geeig- neten Bedingungen erfüllen kann und die Expression der Nukleinsäuresequenz (d.h. Transkription und gegebenenfalls Translation) erfolgt. "Geeignete Bedingungen" meint dabei bevorzugt das Vorliegen der Expressionskassette in einer pflanzlichen Zelle, bevorzugt einer pflanzlichen Zelle umfasst von einem Blütengewebe einer Pflanze.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.
Bevorzugt sind Anordnungen, in denen die transgen zu exprimierende Nukleinsäuresequenz hinter einem der erfindunsggemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12) positioniert wird, so dass beide Sequenzen kovalent miteinander verbunden sind. Bevorzugt ist dabei der Abstand zwischen der Promotorsequenz und der transgen zu exprimierende Nukleinsäuresequenz geringer als 200 Basenpaare, besonders bevorzugt kleiner als 100 Basenpaare, ganz besonders bevorzugt kleiner als 50 Basenpaare.Preference is given to arrangements in which the nucleic acid sequence to be transgenically expressed is positioned behind 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) so that both sequences are covalently linked together. In this case, 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.
Die Herstellung einer funktionellen Verknüpfung als auch die Herstellung eines transgenen Expressionskonstruktes kann mittels gängiger Rekombinations- und Klonie- rungstechniken realisiert werden, wie sie beispielsweise in Maniatis T, Fritsch EF und Sambrook J (1989) Molecular Cloning: A Laboratory Manual, CoId Spring Harbor Labo- ratory, CoId Spring Harbor (NY), in Silhavy TJ, Berman ML und Enquist LW (1984) Experiments with Gene Fusions, CoId Spring Harbor Laboratory, CoId Spring Harbor (NY) und in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience beschrieben sind. Zwischen beide Sequenzen können aber auch weitere Sequenzen positioniert werden, die zum Beispiel die Funktion eines Linkers mit bestimmten Restriktionsenzymschnittstellen oder eines Si- gnalpeptides haben. Auch kann die Insertion von Sequenzen zur Expression von Fusionsproteinen führen. Bevorzugt kann das transgene Expressionskonstrukt, bestehend aus einer Verknüpfung von Promotor und zu exprimierender Nukleinsäuresequenz, integriert in einem Vektor vorliegen und durch zum Beispiel Transformation in ein pflanzliches Genom insertiert werden.
Unter einer Expressionskassette sind aber auch solche Konstruktionen zu verstehen, bei denen einer der erfindungsgemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12), ohne dass er zuvor notwendigerweise mit einer zu exprimierenden Nukleinsäuresequenz funktionell verknüpft wurde, zum Beispiel über eine gezielte homologe Rekombination oder eine zufällige Insertion in ein Wirtsgenom eingeführt wird, dort regulatorische Kontrolle über mit ihm dann funktionell verknüpfte endogene Nukleinsäuresequenzen übernimmt und die transgene Expression derselben steuert. Durch Insertion des Promotors - zum Beispiel durch eine homo- löge Rekombination - vor eine für ein bestimmtes Polypeptid kodierende Nukleinsäure erhält man eine erfindungsgemäße Expressionskassette, die die Expression des bestimmten Polypeptides selektiv in den Geweben der Blüte steuert. Auch kann beispielsweise der natürliche Promotor eines endogenen Gens gegen einen der erfindungsgemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12) ausgetauscht und so das Expressionsverhalten des endogenen Gens modifiziert werden.The production of a functional linkage as well as the production of a transgenic expression construct can be realized by conventional recombination and cloning techniques, as described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, ColD Spring Harbor Labo Spring, Co. Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, CoId Spring Harbor Laboratory, CoId Spring Harbor (NY) and in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience. However, further sequences can also be positioned between the two sequences which, for example, have the function of a linker with particular restriction enzyme sites or a signal peptide. Also, insertion of sequences may result in the expression of fusion proteins. Preferably, the 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. However, 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. Also, for example, 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.
Ferner kann die Insertion des Promotors auch derart erfolgen, dass antisense-RNA zu der für ein bestimmtes Polypeptid kodierenden Nukleinsäure exprimiert wird. Damit wird selektiv die Expression des bestimmten Polypeptides in den Organen der Blüte herunter reguliert oder ausgeschaltet.Furthermore, the insertion of the promoter may also be such that antisense RNA is expressed to the nucleic acid encoding a particular polypeptide. Thus, the expression of the particular polypeptide in the organs of flowering is selectively down-regulated or eliminated.
Analog kann auch eine transgen zu exprimierende Nukleinsäuresequenz - zum Beispiel durch eine homologe Rekombination - hinter die Sequenz kodierend für einen der erfindungsgemäßen Promotoren (z.B. beschrieben durch SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 bzw. 12), die sich in ihrem natürlichen chromosomalen Kontext befindet, so platziert werden, dass man eine erfindungsgemäße Expressionskassette erhält, die die Expression der transgen zu exprimierenden Nukleinsäureseuquenz in den Blütengeweben steuert.Similarly, a 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), which is in its natural chromosomal context, are placed so as to obtain an expression cassette according to the invention which controls the expression of the transgenic nucleic acid sequence to be expressed in the flower tissues.
Die erfindungsgemäßen transgenen Expressionskassetten können weitere genetische Kontrollsequenzen umfassen. Der Begriff der genetischen Kontrollsequenzen ist breit zu verstehen und meint all solche Sequenzen, die einen Einfluss auf das Zustandekommen oder die Funktion einer erfindungsgemäßen transgenen Expressionskassette haben. Genetische Kontrollsequenzen modifizieren zum Beispiel die Transkription und Translation in prokaryotischen oder eukaryotischen Organismen. Vorzugsweise umfassen die erfindungsgemäßen transgenen Expressionskassetten 3'-stromabwärts von der jeweiligen transgen zu exprimierenden Nukleinsäuresequenz eine Terminatorsequenz als zusätzliche genetische Kontrollsequenz, sowie gegebenenfalls weitere übli- che regulative Elemente, und zwar jeweils funktionell verknüpft mit der transgen zu exprimierenden Nukleinsäuresequenz.
Genetische Kontrollsequenzen umfassen auch weitere Promotoren, Promotorelemente oder Mini mal promotoren, die die expressionssteuemden Eigenschaften modifizieren können. So kann durch genetische Kontrollsequenzen zum Beispiel die gewebespezifische Expression zusätzlich abhängig von bestimmten Stressfaktoren erfolgen. Ent- sprechende Elemente sind zum Beispiel für Wasserstress, Abscisinsäure (Lam E und Chua NH, J Biol Chem 1991 ; 266(26):17131-17135) und Hitzestress (Schoffl F et al. (1989) Mol Gen Genetics 217(2-3) :246-53) beschrieben.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. Preferably, 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. For example, by means of genetic control sequences, 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).
Es können ferner weitere Promotoren funktionell mit der zu exprimierenden Nuklein- säuresequenz verknüpft sein, die eine transgene Expression in weiteren Pflanzengeweben oder in anderen Organismen, wie zum Beispiel E.coli Bakterien ermöglichen. Als Promotoren kommen im Prinzip alle pflanzenspezifischen Promotoren in Frage. Pflanzenspezifische Promotoren meint grundsätzlich jeden Promotor, der die Expression von Genen, insbesondere Fremdgenen, in Pflanzen oder Pflanzenteilen, -zellen, - geweben, -kulturen steuern kann. Dabei kann die Expression beispielsweise konstitutiv, induzierbar oder entwicklungsabhängig sein. Bevorzugt sind konstitutive Promotoren, gewebespezifische Promotoren, entwicklungsabhängige Promotoren, chemisch-induzierbare stress-induzierbare oder pathogen-induzierbare Promotoren. Entsprechende Promotoren sind dem Fachmann allgemein bekannt.Furthermore, further 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. Preferred are constitutive promoters, tissue-specific promoters, development-dependent promoters, chemically-inducible stress-inducible or pathogen-inducible promoters. Corresponding promoters are generally known to the person skilled in the art.
Weitere vorteilhafte Kontrollsequenzen sind beispielsweise in den Promotoren grampositiver Bakterien wie amy und SPO2 oder in den Hefe- oder Pilzpromotoren ADC1, MFa, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH zu finden.Further advantageous 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.
Prinzipiell können alle natürlichen Promotoren mit ihren Regulationssequenzen wie die oben genannten für das erfindungsgemäße Verfahren verwendet werden. Darüberhi- naus können auch synthetische Promotoren vorteilhaft verwendet werden.In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used to advantage.
Genetische Kontrollsequenzen umfassen ferner auch die 5'-untranslatierten Regionen, Introns oder nichtkodierende 3'-Region von Genen wie beipielsweise das Actin-1 In- tron, oder die Adh1-S Introns 1 , 2 und 6 (allgemein: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)), bevorzugt der Gene mit dem Genlocus At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110 aus Arabidopsis thaliana. Es kann gezeigt werden, dass derartige Regio- nen eine signifikante Funktion bei der Regulation der Genexpression spielen können. So wurde gezeigt, dass 5'-untranslatierte Sequenzen die transiente Expression hetero- loger Gene verstärken können. Beispielhaft für Translationsverstärker sei die 5'- Leadersequenz aus dem Tabak-Mosaik-Virus zu nennen (GaIMe et al. (1987) Nucl A- cids Res 15:8693-8711) und dergleichen. Sie können ferner die Gewebsspezifität för- dem (Rouster J et al. (1998) Plant J 15:435-440). Die unter SEQ ID NO: 1 , 4, 7, 10, 11 und 12 angegebenen Nukleinsäuresequenzen repräsentieren jeweils die Promotorregion und die 5'-untranslatierte Regionen bis vor das ATG-Startcodon der jeweiligen
Gene mit dem Genlocus At5g33370, At5g22430, At1g26630, At4g35100, At3g04290 und At5g46110.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.
Das transgene Expressionskonstrukt kann vorteilhafterweise eine oder mehrere soge- nannte "enhancer Sequenzen" funktionell verknüpft mit dem Promotor enthalten, die eine erhöhte transgene Expression der Nukleinsäuresequenz ermöglichen. Auch am 3'-Ende der transgen zu exprimierenden Nukleinsäuresequenzen können zusätzliche vorteilhafte Sequenzen inseriert werden, wie weitere regulatorische Elemente oder Terminatoren. Die transgen zu exprimierenden Nukleinsäuresequenzen können in ei- ner oder mehreren Kopien im Genkonstrukt enthalten sein.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.
Als Kontrollsequenzen geeignete Polyadenylierungssignale sind pflanzliche Polyadeny- lierungssignale, vorzugsweise solche, die im wesentlichen T-DNA Polyadenylierungssignale aus Agrobakterium tumefaciens. Beispiele für besonders geeignete Termina- torsequenzen sind der OCS (Octopin-Synthase)-Terminator und der NOS (Nopalin- Synthase)-Terminator.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.
Als Kontrollsequenzen sind weiterhin solche zu verstehen, die eine homologe Rekombination bzw. Insertion in das Genom eines Wirtsorganismus ermöglichen oder die Entfernung aus dem Genom erlauben. Bei der homologen Rekombination kann zum Beispiel die kodierende Sequenz eines bestimmten endogenen Gens gegen die für eine dsRNA kodierende Sequenz gezielt ausgetauscht werden. Methoden wie die cre/lox-Technologie erlauben eine gewebespezifische, unter Umständen induzierbare Entfernung des transgenen Expressionskonstruktes aus dem Genom des Wirtsorga- nismus (Sauer B (1998) Methods 14(4):381-92). Hier werden bestimmte flankierende Sequenzen dem Zielgen angefügt (lox-Sequenzen), die später eine Entfernung mittels der cre-Rekombinase ermöglichen.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. In homologous recombination, for example, 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). Here, certain flanking sequences are added to the target gene (lox sequences), which later enable removal by means of the cre recombinase.
Eine transgene Expressionskassette und/oder die von ihm abgeleiteten transgenen Expressionsvektoren können weitere Funktionselemente enthalten. Der Begriff Funktionselement ist breit zu verstehen und meint all solche Elemente, die einen Einfluss auf Herstellung, Vermehrung oder Funktion der erfindungsgemäßen transgenen Expressi- onskonstrukte, der transgenen Expressionsvektoren oder der transgenen Organismen haben. Beispielhaft aber nicht einschränkend seien zu nennen: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. By way of example but not by way of limitation:
a) Selektionsmarker, die eine Resistenz gegen Biozide wie Metabolismusinhibitoren (z.B. 2-Desoxyglucose-6-phosphat; WO 98/45456), Antibiotika (z.B. Kanamycin, G 418, Bleomycin, Hygromycin) oder - bevorzugt - Herbizide (z.B. Phosphinotri- cin) verleihen. Als Selektionsmarker seien beispielhaft genannt: Phosphinothrici- nacetyltransferasen (bar und pat Gen), welche Glutaminsynthaseinhibitoren inaktivieren, 5-Enolpyruvylshikimat-3-phosphatsynthasen (EPSP Synthasegene), die eine Resistenz gegen Glyphosatr (N-(phosphonomethyl)glycin) verleihen,
Glyphosatr degradierende Enzyme (gox-Genprodukt; Glyphosatoxidoreduktase), Dehalogenasen, welche z.B. Dalapon inaktivieren (deh Genprodukt), Sulfonylu- rea- und Imidazolinon inaktivierende Acetolactatsynthasen sowie Nitrilasen, welche z.B. Bromoxynil degradieren (bxn Genprodukt), das aasa-Genprodukt, das eine Resistenz gegen das Antibiotikum Apectinomycin verleih, Streptomy- cinphosphotransferasen (SPT), die eine Resistenz gegen Streptomycin gewähren, Neomycinphosphotransferasen (NPTII), die eine Resistenz gegen Kanamycin oder Geneticidin verleihen, das Hygromycinphosphotransferasen (HPT), die eine Resistenz gegen Hygromycin vermitteln, das Acetolactatsynthasen (ALS), die ei- ne Resistenz gegen Sulfonylhamstoff-Herbizide verleihen (z.B. mutierte ALS-a) Selection markers which are resistant to biocides such as metabolism inhibitors (eg 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (eg kanamycin, G 418, bleomycin, hygromycin) or - preferably - herbicides (eg phosphinotriquin) to lend. Examples of 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 phosphotransferases (NPTII) that confer resistance to kanamycin or geneticididine, hygromycin phosphotransferases (HPTs) that confer resistance to hygromycin, acetolactate synthases ( ALS) conferring resistance to sulfonylurea herbicides (eg mutated ALS)
Varianten mit z.B. der S4 und/oder Hra Mutation).Variants with e.g. the S4 and / or Hra mutation).
b) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Transformationseffizienz oder des Expressionsortes oder -Zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Reporter-Proteine (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) wie das "green fluorescence protein" (GFP) (Sheen et al.(1995) Plant Journal 8(5):777-784), die Chloramphenicoltransferase, eine Luziferase (Ow et al. (1986) Science 234:856-859), das Aequorin-Gen (Prasher et al. (1985) Bio- ehern Biophys Res Commun 126(3): 1259-1268), die b-Galactosidase, ganz besonders bevorzugt ist die ß-Glucuronidase (Jefferson et al. (1987) EMBO J 6:3901-3907).b) 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. (1985) Biophys Biophys Res Commun 126 (1985); 3): 1259-1268), the b-galactosidase, most preferably the β-glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
c) Replikationsursprünge, die eine Vermehrung der erfindungsgemäßen transgenen Expressionskonstrukte oder transgenen Expressionsvektoren in zum Beispielc) origins of replication comprising an increase of the transgenic expression constructs or transgenic expression vectors according to the invention in, for example,
E.coli gewährleisten. Beispielhaft seien genannt ORI (origin of DNA replication), der pBR322 ori oder der P15A ori (Sambrook et al.: Molecular Cloning. A Labora- tory Manual, 2nd ed. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY, 1989).Ensure 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 ).
d) Elemente, die für eine Agrobakterium vermittelte Pflanzentransformation erforderlich sind, wie zum Beispiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.d) Elements required for Agrobacterium-mediated plant transformation, such as the right or left border of the T-DNA or the vir region.
"Einführen" umfasst im Rahmen der Erfindung alle Verfahren, die dazu geeignet sind, eine Nukleinsäuresequenz (beispielsweise eine erfindungsgemäße Expressionskassette) direkt oder indirekt, in einen Organismus (z.B. ein Pflanze) oder eine Zelle, Kompar- timent, Gewebe, Organ oder Vermehrungsmaterial (z.B. Samen oder FRüchte) derselben einzuführen oder dort zu generieren. Direkte und indirekte Verfahren sind umfasst. Das Einbringen kann zu einer vorübergehenden (transienten) Präsenz besagter Nukleinsäuresequenz führen oder aber auch zu einer dauerhaften (stabilen). Einführen umfasst beispielsweise Verfahren wie Transfektion, Transduktion oder Transformation.
Die in den Verfahren verwendeten Organismen werden je nach Wirtsorganismus in dem Fachmann bekannter Weise angezogen bzw. gezüchtet."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, compartiment, tissue, organ or propagation material ( Eg seeds or FRüchte) 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.
Das Einführen einer erfindungsgemässen transgenen Expressionskassette in einen Organismus oder Zellen, Geweben, Organe, Teile bzw. Samen desselben (bevorzugt in Pflanzen bzw. pflanzliche Zellen, Gewebe, Organe, Teile oder Samen) kann vorteilhaft unter Verwendung von Vektoren realisiert werden, in denen die transgenen Expressionskassetten enthalten sind. Vektoren können beispielhaft Plasmide, Cosmide, Phagen, Viren oder auch Agrobakterien sein. Die transgenen Expressionskassetten können in den Vektor (bevorzugt ein Plasmidvektor) über eine geeignete Restriktionsschnittstelle insertiert werden. Der entstandene Vektor kann zunächst in E.coli eingeführt und amplifiziert werden. Korrekt transformierte E.coli werden selektioniert, gezüchtet und der rekombinante Vektor mit dem Fachmann geläufigen Methoden gewonnen. Restriktionsanalyse und Sequenzierung können dazu dienen, den Klonie- rungsschritt zu überprüfen. Bevorzugt sind solche Vektoren, die eine stabile Integration der Expressionskassette in das Wirtsgenom ermöglichen.The introduction of a transgenic expression cassette 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.
Die Herstellung eines transformierten Organismus (bzw. einer transformierten Zelle oder Gewebes) erfordert, dass die entsprechende DNA (z.B. der Expressionsvektor) oder RNA in die entsprechende Wirtszelle eingebracht wird. Für diesen Vorgang, der als Transformation (oder Transduktion bzw. Transfektion) bezeichnet wird, steht eine Vielzahl von Methoden zur Verfügung (Keown et al. (1990) Methods in Enzymology 185:527-537). So kann die DNA oder RNA beispielhaft direkt durch Mikroinjektion oder durch Bombardierung mit DNA-beschichteten Mikropartikeln eingeführt werden. Auch kann die Zelle chemisch, zum Beispiel mit Polyethylenglycol, permeabilisiert werden, so dass die DNA durch Diffusion in die Zelle gelangen kann. Die DNA kann auch durch Protoplastenfusion mit anderen DNA-enthaltenden Einheiten wie Minicells, Zellen, Ly- sosomen oder Liposomen erfolgen. Elektroporation ist eine weitere geeignete Methode zum Einführen von DNA, bei der die Zellen reversibel durch einen elektrischen Impuls permeabilisert werden. Entsprechende Verfahren sind beschrieben (beispielsweise bei Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104- 112; Guerche et al. (1987) Plant Science 52:111-116; 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. (1989) Plant Physiology 91 :694-701 ; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).The production of a transformed organism (or a transformed cell or tissue) requires that the appropriate DNA (e.g., the expression vector) or RNA be introduced into the appropriate host cell. For this process, referred to as transformation (or transfection), a variety of methods are available (Keown et al., (1990) Methods in Enzymology 185: 527-537). For example, the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. Also, 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. (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).
Als Vektoren zur Expression in E.coli sind bevorzugt pQE70, pQE60 und pQE-9 (QIAGEN, Inc.); pBluescript Vektoren, Phagescript Vektoren, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).
Bevorzugte Vektoren zur Expression in Säugerzellen umfassen pWLNEO, pSV2CAT, pOG44, pXT1 und pSG (Stratagene Inc.); pSVK3, pBPV, pMSG und pSVL (Pharmacia Biotech, Inc.). Als induzierbare Vektoren seien 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.) oder die pMAM-Serie (Clontech, Inc.; GenBank Accession No.: U02443) zunennen. Diese stellen bereits das induzierbare regulatorische Kontrollelement beispielsweise für eine chemisch, induzierbare Expression zur Verfügung.As 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.). As 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.
Vektoren für die Expression in Hefe umfassen beispielhaft pYES2, pYD1 , pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2, PHIL-SI, pPIC3SK, pPIC9K, und PA0815 (Invitrogen, Inc.).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 .).
Klonierungsvektoren und Techniken zur genetischen Manipulation von Ciliaten und Algen sind dem Fachmann bekannt (WO 98/01572; Falciatore et al. (1999) Marine Bio- technology 1(3):239-251; Dunahay et al. (1995) J Phycol 31 :10004-1012).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).
Prinzipiell sind für die Transformation tierischer Zellen oder von Hefezellen ähnliche Verfahren wie für die "direkte" Tranformation von pflanzlichen Zellen anzuwenden. Insbesondere Verfahren wie die Calciumphosphat oder Liposomen vermittelte Transformation oder aber Elektroporation sind bevorzugt.In principle, similar procedures are used for the transformation of animal cells or yeast cells as for the "direct" transformation of plant cells. In particular, methods such as calcium phosphate or liposome mediated transformation or electroporation are preferred.
Verschiedene Methoden und Vektoren zum Einschleusen von Genen in das Genom von Pflanzen sowie zur Regeneration von Pflanzen aus Pflanzengeweben oder Pflanzenzellen sind bekannt (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Kapitel 6/7, S. 71-119 (1993); White FF (1993) Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung and Wu R, Academic Press, 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung and R. Wu, Academic Press, S.128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; Haiford NG, Shewry PR (2000) Br Med Bull 56(1):62-73). Dazu zählen beispielhaft die oben erwähnten. Bei Pflanzen werden dabei die beschriebenen Methoden zur Transformation und Regeneration von Pflanzen aus Pflanzengeweben oder Pflanzenzellen zur transienten oder stabilen Transformation genutzt. Geeignete Methoden sind vor allem die Protoplastentransformation durch Polyethylenglykol- induzierte DNA-Aufnahme, Calciumphosphat-vermittelte Transformation, DEAE- Dextran-vermittelte Transformation, Liposomen vermittelte Transformation (Freeman et al. (1984) Plant Cell Physiol. 29:1353ff; US 4,536,475), biolistische Verfahren mit der Genkanone ("particle bombardment" Methode; 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. (1990) Plant Cell 2:603), die Elektroporation, die Inkubation trockener Embryonen in
DNA-haltiger Lösung, Elektroporation (EP-A 290 395, WO 87/06614), Mikroinjektion (WO 92/09696, WO 94/00583, EP-A 0 331 083, EP-A 0 175 966) oder andere Methoden der direkten DNA-Einführung (DE 4 005 152, WO 90/12096, US 4,684,611). Physikalische Methoden der DNA-Einführung in pflanzliche Zellen sind im Überblick darge- stellt bei Oard (1991) Biotech Adv 9:1-11.Various methods and vectors for introducing genes into the genome of plants and for regenerating plants from plant tissues or plant cells are known (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993), White FF (1993) Vectors for Gene Transfer to Higher Plants, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and Wu R, Academic Press, 15-38, Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, pp. 128-143, Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225, Haiford NG, Shewry PR (2000) Br Med Bull 56 (1): 62-73). These include, by way of example, the ones mentioned above. In plants, the methods described for the transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation. 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. (1990) Plant Cell 2: 603), electroporation, the incubation of dry embryos in DNA-containing solution, electroporation (EP-A 290 395, WO 87/06614), microinjection (WO 92/09696, WO 94/00583, EP-A 0 331 083, EP-A 0 175 966) or other methods of direct DNA introduction (DE 4 005 152, WO 90/12096, US 4,684,611). Physical methods of introducing DNA into plant cells are reviewed in Oard (1991) Biotech Adv 9: 1-11.
Im Falle dieser "direkten" Transformationsmethoden sind keine besonderen Anforderungen an das verwendete Plasmid gestellt. Einfache Plasmide wie die der pUC-Reihe, pBR322, M13mp Reihe, pACYC184 etc. können verwendet werden. Sollen vollständi- ge Pflanzen aus den transformierten Zellen regeneriert werden, so ist es erforderlich, dass sich auf dem Plasmid ein zusätzliches selektionierbares Markergen befindet.In the case of these "direct" transformation methods, no special requirements are placed on the plasmid used. Simple plasmids such as the pUC series, pBR322, M13mp series, pACYC184, etc. can be used. If complete plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be present on the plasmid.
Neben diesen "direkten" Transformationstechniken kann eine Transformation auch durch bakterielle Infektion mittels Agrobakterium (z.B. EP 0 116 718), virale Infektion mittels viraler Vektoren (EP 0 067 553; US 4,407,956; WO 95/34668; WO 93/03161) oder mittels Pollen (EP 0 270 356; WO 85/01856; US 4,684,611) durchgeführt werden.In addition to these "direct" transformation techniques, 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).
Bevorzugt erfolgt die Transformation mittels Agrobakterien, die "entwaffnete" (disar- med) Ti-Plasmidvektoren enthalten, wobei deren natürliche Fahrigkeit zum Gentransfer auf Pflanzen genutzt wird (EP-A 0 270 355; EP-A 0 116 718).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).
Agrobakterium-Transformation ist weit verbreitet für die Transformation von Dicotyle- donen, wird aber auch zunehmend auf Monocotyledonen angewandt (Toriyama et al. (1988) Bio/Technology 6: 1072-1074; Zhang et al. (1988) Plant Cell Rep 7:379-384; 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. (1992) Plant Cell Rep 11 :585-591 ; Li et al. (1993) Plant Cell Rep 12:250-255; Rathore et al. (1993) Plant Mol Biol 21 :871-884; Fromm et al. (1990) Bio/Technology 8:833-839; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; D'Halluin et al. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant Mol Biol 18:189-200; Koziel et al. (1993) Biotechnology 11 :194-200; Vasil IK (1994) Plant Mol Biol 25:925-937; Weeks et al. (1993) Plant Physiol 102:1077-1084; Somers et al. (1992) Bio/Technology 10:1589-1594; WO 92/14828; Hiei et al. (1994) Plant J 6:271-282).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. (1992) Plant Cell Rep 11: 585-591 Li et al (1993) Plant Cell Rep 12: 250-255; Rathore et al. (1993) Plant Mol Biol 21: 871-884; Fromm et al. (1990) Bio / Technology 8: 833-839; Gordon -Kamm et al. (1990) Plant Cell 2: 603-618; D'Halluin et al (1992) Plant Cell 4: 1495-1505; Walters et al. (1992) Plant Mol Biol 18: 189-200; et al. (1993) Biotechnology 11: 194-200; Vasil IK (1994) Plant Mol Biol 25: 925-937; Weeks et al. (1993) Plant Physiol 102: 1077-1084; ers et al. (1992) Bio / Technology 10: 1589-1594; WO 92/14828; Hiei et al. (1994) Plant J 6: 271-282).
Die für die Agrobakterium-Transformation meist verwendeten Stämme Agrobakterium tumefaciens oder Agrobakterium rhizogenes enthalten ein Plasmid (Ti bzw. Ri Plasmid), das auf die Pflanze nach Agrobakterium-Infektion übertragen wird. Ein Teil dieses Plasmids, genannt T-DNA (transferred DNA), wird in das Genom der Pflanzenzelle integriert. Alternativ können durch Agrobakterium auch binäre Vektoren (Mini-Ti- Plasmide) auf pflanzen übertragen und in deren Genom integriert werden.
Die Anwendung von Agrobakterium tumefaciens für die Transformation von Pflanzen unter Verwendung von Gewebekulturexplantaten ist beschrieben (u.a. Horsch RB et al. (1985) Science 225:1229ff.; Fraley et al. (1983) Proc Natl Acad Sei USA 80: 4803- 4807; Bevans et al. (1983) Nature 304:184-187). Viele Stämme von Agrobakterium tumefaciens sind in der Lage, genetisches Material - beispielsweise die erfindungsgemäßen Expressionskassetten - zu übertragen, wie z.B. die Stämme EHA101[pEHA101], EHA105[pEHA105], LBA4404[pAL4404], C58C1 [pMP90] und C58C1 [pGV2260] (Hood et al. (1993) Transgenic Res 2:208-218; Hoekema et al. (1983) Nature 303:179-181; Koncz and Schell (1986) Gen Genet 204:383-396; Deblae- re et al. (1985) Nucl Acids Res 13: 4777-4788).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. Alternatively, Agrobacterium also allows binary vectors (mini-Ti plasmids) to be transferred to plants and integrated into their genome. The use of 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). Many 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).
Werden Agrobakterien verwendet, so ist die Expressionskassette in spezielle Plasmide zu integrieren, entweder in einen Zwischenvektor (englisch: Shuttle or intermediate vector) oder einen binären Vektor. Bevorzugt werden binäre Vektoren verwendet, die sowohl in E.coli als auch in Agrobakterium replizieren können. Sie enthalten in der Regel ein Selektionsmarkergen und einen Linker oder Polylinker, flankiert von der rechten und linken T-DNA Begrenzungssequenz. Sie können direkt in Agrobakterium transformiert werden (Holsters et al. (1978) Mol Gen Genet 163:181-187). Das in diesem Fall als Wirtsorganismus fungierende Agrobakterium sollte bereits ein Plasmid mit der vir- Region enthalten. Diese ist für die Übertragung der T-DNA auf die pflanzliche Zelle erforderlich. Ein so transformiertes Agrobakterium kann zur Transformation pflanzlicher Zellen verwendet werden. Die Verwendung von T-DNA zur Transformation pflanzlicher Zellen ist intensiv untersucht und beschrieben (EP-A 0 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287). Verschiedene binäre Vektoren sind bekannt und teilweise kommerziell erhältlich wie zum Beispiel pBI101.2 oder pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al.(1984) Nucl Acids Res 12:8711), pBinAR, pPZP200 oder pPTV.If Agrobacteria are used, then the expression cassette is to be integrated into special plasmids, either into an intermediate vector (shuttle or intermediate vector) or a binary vector. Preference is given to using 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. The use of 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.
Die mit einem solchen Vektor transformierten Agrobakterien können dann in bekannter Weise zur Transformation von Pflanzen, insbesondere von Kulturpflanzen, wie z.B. von Raps, verwendet werden, indem beispielsweise verwundete Blätter oder Blattstücke in einer Agrobakterienlösung gebadet und anschliessend in geeigneten Medien kultiviert werden. Die Transformation von Pflanzen durch Agrobakterien ist beschrieben (White FF (1993) Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, herausgegeben von SD Kung and R Wu, Academic Press, S. 15-38; Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, herausgegeben von S.D. Kung and R. Wu, Acade- mic Press, S.128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec BiolThe 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
42:205-225). Aus den transformierten Zellen der verwundeten Blätter bzw. Blattstücke
können in bekannter Weise transgene Pflanzen regeneriert werden, die integriert die oben beschriebenen erfindungsgemässen Expressionssysteme enthalten.42: 205-225). From the transformed cells of the wounded leaves or leaf pieces For example, it is possible in a known manner to regenerate transgenic plants which contain the above-described expression systems according to the invention in an integrated manner.
Stabil transformierte Zellen (d.h. solche, die die eingeführte DNA integriert in die DNA der Wirtszelle enthalten) können von untransformierten selektioniert werden, wenn ein selektionierbarer Marker Bestandteil der eingeführten DNA ist. Als Marker kann beispielhaft jedes Gen fungieren, dass eine Resistenz gegen ein Biozid (z.B. ein Antibiotikum oder Herbizid (s.o.) zu verleihen vermag (s.o.). Transformierte Zellen, die ein solches Markergen exprimieren, sind in der Lage, in der Gegenwart von Konzentrationen eines entsprechenden Biozids zu überleben, die einen untransformierten Wildtyp abtöten. Der Selektionsmarker erlaubt die Selektion von transformierten Zellen von untransformierten (McCormick et al. (1986) Plant Cell Reports 5:81-84). Die erhaltenen Pflanzen können in üblicher Weise gezüchtet und gekreuzt werden. Zwei oder mehr Generationen sollten kultiviert werden, um sicherzustellen, dass die genomische Integration stabil und vererblich ist.Stably transformed cells (i.e., those containing the introduced DNA integrated into the DNA of the host cell) can be selected from untransformed if a selectable marker is part of the introduced DNA. By way of example, any gene capable of conferring resistance to a biocide (eg, an antibiotic or herbicide, etc.) may be used as a marker 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.
Sobald eine transformierte Pflanzenzelle hergestellt wurde, kann eine vollständige Pflanze unter Verwendung von dem Fachmann bekannten Verfahren erhalten werden. Hierbei geht man beispielhaft von Kalluskulturen, einzelnen Zellen (z.B. Protoplasten) oder Blattscheiben aus (Vasil et al. (1984) Cell Culture and Somatic CeI Genetics of Plants, VoI I, Il and IM, Laboratory Procedures and Their Applications, Academic Press; Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press). Aus diesen noch undifferenzierten Kallus-Zellmassen kann die Bildung von Spross und Wurzel in bekannter Weise induziert werden. Die erhaltenen Sprösslinge können ausgepflanzt und gezüchtet werden. Entsprechende Verfahren sind beschrieben (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).Once a transformed plant cell has been produced, 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).
Die Wirksamkeit der Expression der transgen exprimierten Nukleinsäuren kann bei- spielsweise in vitro durch Sprossmeristemvermehrung unter Verwendung einer der oben beschriebenen Selektionsmethoden ermittelt werden. Zudem kann eine in Art und Höhe veränderte Expression eines Zielgens und die Auswirkung auf den Phänptyp der Pflanze an Testpflanzen in Gewächshausversuchen getestet werden.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. In addition, 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.
Ein weiterer Gegenstand der Erfindung betrifft transgene Organismen, transformiert mit wenigstens einer erfindungsgemässen Expressionskassette oder einem erfindungsgemässen Vektor, sowie Zellen, Zellkulturen, Gewebe, Teile - wie zum Beispiel bei pflanzlichen Organismen Blätter, Wurzeln usw.- oder Vermehrungsgut abgeleitet von solchen Organismen.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.
Unter Organismus, Ausgangs- oder Wirtsorganismen werden prokaryotische oder eu- karyotische Organismen, wie beispielsweise Mikroorganismen oder pflanzliche Orga-
nismen verstanden. Bevorzugte Mikroorganismen sind Bakterien, Hefen, Algen oder Pilze.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.
Bevorzugte Bakterien sind Bakterien der Gattung Escherichia, Erwinia, Agrobakterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus oder Cyanobakterien zum Beispiel der Gattung Synechocystis und weitere in Brock Biology of Microorganisms Eighth Edition auf den Seiten A-8, A-9, A10 und A11 beschriebenen Bakteriengattungen.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.
Bevorzugt sind vor allem Mikroorganismen, welche zur Infektion von Pflanzen und damit zur Übertragung der erfindungsgemässen Konstrukte befähigt sind. Bevorzugte Mikroorganismus sind solche aus der Gattung Agrobakterium und insbesondere der Art Agrobakterium turnefaciens. Besonders bevorzugte Mikroorganismen sind solche, die zur Produktion von Toxinen (z.B. Botulinus Toxin), Pigmenten (z.B. Carotinoiden oder Flavonoiden), Antibiotika (z.B. Penicillin), Phenylpropanoiden (z.B. Tocopherol), Polyungesättigten Fettsäuren (z.B.Arachidonsäure) oder Vitaminen (z.B. Vitamin B12) befähigt sind.Preference is given above all to 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.
Bevorzugte Hefen sind Candida, Saccharomyces, Hansenula, Phaffia rhodozyma oder Pichia.Preferred yeasts are Candida, Saccharomyces, Hansenula, Phaffia rhodozyma or Pichia.
Bevorzugte Pilze sind Aspergillus, Trichoderma, Blakeslea, Ashbya, Neurospora, Fusa- rium, Beauveria oder weitere in Indian Chem Engr. Section B. VoI 37, No 1 ,2 (1995) auf Seite 15, Tabelle 6 beschriebene Pilze.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.
Als transgene Organismen bevorzugte Wirts- oder Ausgangsorganismen sind vor allem pflanzliche Organismen.Preferred transgenic organisms host or initial organisms are mainly plant organisms.
"Pflanzlicher Organismus oder von diesem abgeleitete Zellen" meint allgemein jede Zelle, Gewebe, Teile oder Vermehrungsgut (wie Samen oder Früchte) eines zur Photosynthese befähigten Organismus. Eingeschlossen sind im Rahmen der Erfindung alle Gattungen und Arten höherer und niederer Pflanzen des Pflanzenreiches. Einjährige, mehrjährige, monocotyledone und dicotyledone Pflanzen sind bevorzugt."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.
"Pflanze" im Rahmen der Erfindung meint alle Gattungen und Arten höherer und niederer Pflanzen des Pflanzenreiches. Eingeschlossen unter dem Begriff sind die reifen Pflanzen, Saatgut, Sprosse und Keimlinge, sowie davon abgeleitete Teile, Vermehrungsgut (zum Beispiel Knollen, Samen oder Früchte), Pflanzenorgane, Gewebe, Protoplasten, Kallus and undere Kulturen, zum Beispiel Zell- oder Kalluskulturen, sowie alle anderen Arten von Gruppierungen von Pflanzenzellen zu funktionellen oder strukturellen Einheiten. Reife Pflanzen meint Pflanzen zu jedem beliebigen Entwicklungs-
Stadium jenseits des Keimlings. Keimling meint eine junge, unreife Pflanze in einem frühen Entwicklungsstadium."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.
Pflanzliche Organismen im Sinne der Erfindung sind weiterhin weitere photosynthe- tisch aktive Organismen, wie zum Beispiel Algen, Cyanobakterien sowie Moose. Bevorzugte Algen sind Grünalgen, wie beispielsweise Algen der Gattung Haematococcus, Phaedactylum tricomatum, Pirellula, Volvox oder Dunaliella. Insbesondere bevorzugt sind Synechocystis, Chlamydomonas und Scenedesmus.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. Especially preferred are Synechocystis, Chlamydomonas and Scenedesmus.
Im Rahmen des erfindungesgemäßen Verfahrens sind insbesondere pflanzliche Organismen bevorzugt ausgewählt aus der Gruppe der Blütenpflanzen (Phylum Anthophyta "Angiospermen"). Umfasst sind alle einjährigen und mehrjährige, monokotyledonen und dikotyledonen Pflanzen. Bevorzugt ist die Pflanze aus nachfolgenden Pflanzenfamilien ausgewählt: Amaranthaceae, Amaryllidaceae, Asteraceae, Berberidaceae .Brassicaceae, Cannabaceae , Caprifoliaceae, Carγophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Fabaceae, Gentianaceae, Geraniaceae, Illia- ceae .Labiatae, Lamiaceae, Leguminosae, Liliaceae, Linaceae, Papaveraceae, Papi- lionoideae, Liliaceae, Linaceae, Malvaceae, Oleaceae, Orchidaceae, Poaceae, Primu- laceae, Ranunculaceae , Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniacea, Theaceae, Tropaeolaceae, Umbelliferae undVitaceae.In the context of the method according to the invention, in particular 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, Tropaeolaceae, Umbelliferae and Vitaceae.
Die Erfindung wird ganz besonders bevorzugt auf dikotyledone pflanzliche Organismen angewendet. Bevorzugte dikotyle Pflanzen sind insbesondere ausgewählt aus den dikotylen Kulturpflanzen, wie zum Beispiel den nachfolgendenThe invention is most preferably applied to dicotyledonous plant organisms. Preferred dicotyledonous plants are especially selected from the dicotyledonous crops, such as the following
1) Kategorie: Dicotyledonae (Dicotyledonen). Bevorzugte Familien:1) Category: Dicotyledonae (dicotyledons). Preferred families:
Aceraceae (Ahornhölzer)Aceraceae (Maple Woods)
Cactaceae (Kakteen)Cactaceae (cacti)
Rosaceae (Rosen, Äpfel, Mandeln, Erdbeeren)Rosaceae (roses, apples, almonds, strawberries)
- Salicaceae (Weiden)- Salicaceae (willows)
Asteraceae (Compositae) besonders die Gattung Lactuca, ganz besonders die Art sativa (Salat), sowie Sonnenblume, Löwenzahn, Tagetes oder Calendula and andere mehr,Asteraceae (Compositae) especially the genus Lactuca, especially the species sativa (lettuce), as well as sunflower, dandelion, Tagetes or Calendula and others more,
Cruciferae (Brassicaceae), besonders die Gattung Brassica, ganz besonders die Arten napus (Raps), campestris (Rübe), oleracea (z.B. Kohl, Blumenkohl
oder Broccoli und weitere Kohlarten); und der Gattung Arabidopsis, ganz besonders die Art thaliana sowie Kresse, Rettich, Canola und andere mehr,Cruciferae (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 wie Melone, Kürbis, Gurken oder Zucchini und andere mehr,Cucurbitaceae such as melon, pumpkin, cucumber or zucchini and more,
Leguminosae (Fabaceae) besonders die Gattung Glycine, ganz besonders die Art max (Sojabohne) Soja sowie Alfalfa, Erbse, Bohnengewächsen, Lupine oder Erdnuss und andere mehr,Leguminosae (Fabaceae) especially the genus Glycine, especially the species max (soybean) soy as well as alfalfa, pea, bean plants, lupine or peanut and others more,
- Malvaceae insbesondere Malve, Baumwolle, eßbarer Eibisch, Hibiscus und andere mehr,- Malvaceae in particular mallow, cotton, edible marshmallow, hibiscus and others more,
Rubiaceae, bevorzugt der Unterklasse Lamiidae wie beispielsweise Coffea arabica oder Coffea liberica (Kaffeestrauch) und andere mehr,Rubiaceae, preferably of the subclass Lamiidae such as Coffea arabica or Coffea liberica (coffee shrub) and others more
Solanaceae besonders die Gattung Lycopersicon, ganz besonders die Art es- culentum (Tomate) und die Gattung Solanum, ganz besonders die Art tuberosum (Kartoffel) und melongena (Aubergine) und die Gattung Capsicum, ganz besonders die Art annum (Paprika), sowie Tabak, Petunie und andere mehr,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, bevorzugt der Unterklasse Dilleniidae wie beispielsweise The- obroma cacao (Kakaostrauch) und andere mehr,Sterculiaceae, preferably of the subclass Dilleniidae such as, for example, The obroma cacao (cocoa bush) and others more
Theaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Camellia sinensis oder Thea sinensis (Teestrauch) und andere mehr,Theaceae, preferably of the subclass Dilleniidae such as Camellia sinensis or Thea sinensis (tea shrubs) and others more
Umbelliferae (Apiaceae), besonders die Gattung Daucus (ganz besonders die Art carota (Karotte)), Apium (ganz besonders die Art graveolens dulce (Sellerie)) sowie Petersilie und andere mehr;Umbelliferae (Apiaceae), especially the genus Daucus (especially the species carota), Apium (especially the species graveolens dulce (celery)) as well as parsley and others;
sowie Lein, Hanf, Flachs, Spinat, Möhre, Zuckerrübe und den verschiedenen Baum-,as well as flax, hemp, flax, spinach, carrot, sugar beet and the various tree,
Nuss- und Weinarten, insbesondere Banane und Kiwi.Nut and wine, especially banana and kiwi.
Darüberhinaus sind jedoch auch monokotyle Pflanzen geeignet. Bevorzugt sind diese ausgewählt aus den monokotylen Kulturpflanzen, wie zum Beispiel den FamilienIn addition, however, monocot plants are also suitable. Preferably, these are selected from the monocotyledonous crops, such as the families
Arecaceae (Palmen)Arecaceae (palm trees)
Bromeliaceae (Ananas, spanisches Moos) Cyperaceae (Seggen) - Liliaceae (Lillien, Tulpen, Hyazinthen, Zwiebel, Knoblauch) Orchidaceae (Orchideen) Poaceae (Gräser, Bambusse, Mais, Zuckerrohr, Weizen)
Iridaceae (Blenden, Gladiolen, Krokusse)Bromeliaceae (pineapple, Spanish moss) Cyperaceae (sedges) - Liliaceae (lillies, tulips, hyacinths, onion, garlic) Orchidaceae (orchids) Poaceae (grasses, bamboos, corn, sugarcane, wheat) Iridaceae (irises, gladioli, crocuses)
Ganz besonders bevorzugt sind Gramineae wie Reis, Mais, Weizen oder andere Getreidearten wie Gerste, Hirse, Roggen, Triticale oder Hafer sowie dem Zuckerrohr so- wie alle Arten von Gräsern.Very particular preference is given to 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.
Ganz besonders bevorzugte Pflanzen sind ausgewählt aus der Gruppe der Pflanzengattungen Mangold, Tagetes errecta, Tagetes patula, Acacia, Aconitum, Adonis, Arni- ca, 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, Gre- villea, Helenium, Helianthus, Hepatica, Heracleum, Hisbiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kenia, Laburnum, Lathyrus, Leontodon, LiIi- um, Linum, Lotus, Lycopersicon, Lysimachia, Maratia, Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia, Photinia, Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus, Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis, Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum, Tulipa, Tussi- lago, Ulex, Viola oder Zinnia, besonders bevorzugt ausgewählt aus der Gruppe der Pflanzengattungen Marigold, Tagetes erecta, Tagetes patula, Lycopersicon, Rosa,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, Linum, Lotus, Lycopersicon, Lysimachia, Maratia, Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia , Photinia, Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus, Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis, Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum, Tulipa, Tussilago, Ul ex, Viola or Zinnia, more preferably selected from the group of plant genera Marigold, Tagetes erecta, Tagetes patula, Lycopersicon, Rosa,
Calendula, Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium, Tropaeolum oder Adonis.Calendula, Physalis, Medicago, Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium, Tropaeolum or Adonis.
Im Rahmen der erfindungsgemässen Expressionskassette kann die Expression einer bestimmten Nukleinsäure durch einen Promotor mit Spezifität für die Organe der Blüte zu Bildung von sense-RNA, antisense RNA oder doppelsträngiger RNA in Form einer inversen Wiederholung (dsRNAi) führen. Die sense-RNA kann infolge in bestimmte Polypeptide translatiert werden. Mit der antisense-RNA und dsRNAi kann die Expression bestimmter Gene herunterreguliert werden.In the context of the expression cassette according to the invention, 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. With the antisense RNA and dsRNAi, the expression of certain genes can be down-regulated.
Das Verfahren der Genregulation mittels doppelsträngiger RNA ("double-stranded RNA interference"; dsRNAi) ist vielfach in tierischen und pflanzlichen Organismen beschrieben (z.B. Matzke MA et al. (2000) Plant Mol Biol 43:401-415; Fire A et al (1998) Nature 391 :806-811 ; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). Auf die in den angegebenen Zitaten beschriebenen Verfahren und Methoden wird ausdrücklich Bezug genommen.The method of gene regulation by means of double-stranded RNA (dsRNAi) has been widely described in animal and plant organisms (eg Matzke MA et al. (2000) Plant Mol Biol 43: 401-415; Fire A et al. 1998) Nature 391: 806-811, WO 99/32619, WO 99/53050, WO 00/68374, WO 00/44914, WO 00/44895, WO 00/49035, WO 00/63364). The methods and methods described in the cited citations are incorporated herein by reference.
Die Spezifität der erfindungsgemässen Expressionskonstrukte und Vektoren für pflanzliche Blüten ist besonders vorteilhaft. Die Blüte hat eine Funktion im Anlocken von Nutzinsekten durch Pigmenteinlagerung oder Synthese flüchtiger Chemikalien.
Oft sind die natürlichen Abwehrmechanismen der Pflanze zum Beispiel gegen Patho- gene unzureichend. Die Einführung fremder Gene aus Pflanzen, Tieren, oder mikrobi- ellen Quellen kann die Abwehr verstärken. Beispiel sind der Schutz gegen Insekten- frass in Tabak durch Expression des Bacillus thuringiensis Endotoxin (Vaeck et al. (1987) Nature 328:33-37) oder der Schutz des Tabaks gegen Pilzbefall durch Expression einer Chitinase aus der Bohne (Broglie et al. (1991) Science 254:1194-1197).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. Often, 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).
Kälteeinbrüche in der Blütezeit führen jedes Jahr zu erheblichen Ernteverlusten. Eine gezielte Expression schützender Proteine gezielt in der Blüteperiode kann einen Schutz gewähren.Cold spells during flowering lead to significant crop losses each year. Targeted expression of protective proteins specifically during the flowering period can provide protection.
Für eine hohe Effizienz solcher gentechnischer Ansätze ist eine konzentrierte Expression der entsprechenden transgen zu exprimierenden Nukleinsäuresequenz vor allem in der äussersten Hülle der Blüte vorteilhaft. Eine konstitutive Expression in der gesam- ten Pflanze kann den Effekt zum Beispiel durch eine Verdünnung in Frage stellen oder das Wachstum der Pflanze bzw. die Qualität des Pflanzenproduktes beeinträchtigen. Außerdem kann es durch eine konstitutive Expression verstärkt zum Abschalten des Transgens kommen ("gene silencing").For a high efficiency of such genetic engineering approaches, 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. In addition, constitutive expression can lead to increased shutting down of the transgene ("gene silencing").
Hierzu sind Promotoren mit Spezifität für die Blüte vorteilhaft. Dem Fachmann ist eine Vielzahl von Proteinen bekannt, deren rekombinante Expression in der Blüte vorteilhaft sind. Ferner sind dem Fachmann eine Vielzahl von Genen bekannt, durch deren Re- primierung oder Ausschaltung mittels Expression einer entsprechenden antisense-RNA ebenfalls vorteilhafte Effekte erreicht werden können. Beispielhaft jedoch nicht ein- schränkend für vorteilhafte Effekte seien zu nennen: Das Erzielen einer Resistenz gegen abiotische Stressfaktoren (Hitze, Kälte, Trockenheit, erhöhte Feuchtigkeit, Umweltgifte, UV-Strahlung) und biotische Stressfaktoren (Pathogene, Viren, Insekten und Krankheiten), die Verbesserung von Nahrungs- oder Futtereigenschaften, die Verbesserung der Wachstumsrate oder des Ertrages, das Erzielen einer längeren oder frühe- ren Blütezeit, die Veränderung oder Verstärkung des Duftes oder der Farbgebung der Blüten. Für die in diesen Anwendungen einsetzbaren Nukleinsäuresequenzen oder Polypeptide seien beispielhaft, aber nicht einschränkend, zu nennen:For this purpose, 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. Furthermore, a variety of 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. For the nucleic acid sequences or polypeptides which can be used in these applications, by way of example, but not by way of limitation:
1. Verbesserter UV-Schutz der pflanzlichen Blüte durch Veränderung der Pigmentie- rung durch Expression bestimmer Polypeptide wie Enyzme oder Regulatoren der1. Improved UV protection of plant flowering by altering pigmentation by expression of specific polypeptides such as enzymes or regulators of the plant
Flavonoidbiosynthese (z.B. Chalconsynthasen, Phenylalaninammoniumlyasen), der DNA-Reparatur (z.B. Photolyasen; Sakamoto A et al.(1998) DNA Seq 9(5- 6):335-40), der Isoprenoidbiosynthese (z.B. Deoxyxylulose-5-phosphatsyntha- sen), der IPP-Synthese oder der Carotinoidbiosynthese (z.B. Phytoensynthasen, Phytoendesaturasen, Lycopincyclasen, Hydroxylasen oder Ketolasen). Bevorzugt sind Nukleinsäuren, die für die Chalconsynthase aus Arabidopsis thaliana (Gen- Bank Acc.-No.: M20308), die 6-4 Photolyase aus Arabidopsis thaliana (GenBank
Acc.-No.:BAB00748) oder das Blaulicht-Photorezeptor/Photolyase-Homolog (PHHI) aus Arabidopsis thaliana (GenBank Acc.-No.: U62549) oder funktionelle Äquivalente derselben kodieren.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 or carotenoid biosynthesis (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. No .: M20308), the Arabidopsis thaliana 6-4 photolyase (GenBank Acc.-No.:BAB00748) or the Arabidopsis thaliana blue light photoreceptor / photolyase homologue (PHHI) (GenBank Acc. No .: U62549) or functional equivalents thereof.
2. Verbesserter Schutz der pflanzlichen Blüte gegen abiotische Stressfaktoren wie Trockenheit, Hitze, oder Kälte zum Beispiel durch Überexpression von dem "anti- freeze"-Polypeptiden (z.B. aus Myoxocephalus Scorpius; WO 00/00512), dem Arabidopsis thaliana Transkriptionsaktivator CBF1 , Glutamatdehydrogenasen (WO 97/12983, WO 98/11240), einem späten Embryogenesegen (LEA) zum Bei- spiel aus Gerste (WO 97/13843), Calcium-abhängigen Proteinkinasegenen (WO2. Improved protection of plant flowering against abiotic stress factors such as drought, heat or cold, for example by overexpression of the "antifreeze" polypeptides (eg from Myoxocephalus Scorpius, WO 00/00512), the Arabidopsis thaliana transcriptional activator CBF1, glutamate dehydrogenases ( WO 97/12983, WO 98/11240), a late embryogenesis gene (LEA), for example from barley (WO 97/13843), calcium-dependent protein kinase genes (WO
98/26045), Calcineurinen (WO 99/05902), Famesyltransferasen (WO 99/06580; Pei ZM et al. (1998) Science 282:287-290), Ferritin (Deak M et al. (1999) Nature Biotechnology 17:192-196), Oxalatoxidase (WO 99/04013; Dunwell JM (1998) Biotechnology and Genetic Engeneering Reviews 15:1-32), DREBIA-Faktor (de- hydration response element B 1A; Kasuga M et al. (1999) Nature Biotechnology98/26045), calcineurins (WO 99/05902), famesyltransferases (WO 99/06580, Pei ZM et al., (1998) Science 282: 287-290), ferritin (Deak M et al. (1999) Nature Biotechnology 17: 192-196), oxalate oxidase (WO99 / 04013, Dunwell JM (1998) Biotechnology and Genetic Engineering Reviews 15: 1-32), DREBIA factor (de- hydration response element B 1A; Kasuga M et al. (1999) Nature Biotechnology
17:276-286), Genen der Mannitol- oder Trehalosesynthese (z.B. Trehalosephos- phatsynthasen; Trehalosephosphatphosphatasen, WO 97/42326); oder durch Inhibition von Genen wie der Trehalase (WO 97/50561). Besonders bevorzugt sind Nukleinsäuren, die für den transkriptioneilen Aktivator CBFI aus Arabidopsis tha- Nana (Gen-Bank Acc.-No.: U77378) oder das "antifreeze"-Protein" aus Myoxocephalus octodecemspinosus (GenBank Acc.-No.: AF306348) oder funktionelle Äquivalente derselben kodieren.17: 276-286), 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). Particular preference is given to 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.
3. Erreichen einer Resistenz zum Beispiel gegen Pilze, Insekten, Nematoden und Krankheiten durch gezielte Absonderung oder Anreicherung bestimmter Metaboli- ten oder Proteine in der Blüte. Beispielhaft seien genannt Glucosinolate (Nemato- denabwehr), Chitinasen oder Glucanasen und andere Enzyme, die die Zellwand von Parasiten zerstören, Ribosom-inaktivierende Proteine (RIPs) und andere Proteine der pflanzlichen Resistenz- und Stressreaktion, wie sie bei Verletzung oder mikrobiellen Befall von Pflanzen oder chemisch durch zum Beispiel Salicylsäure,3. Achieving resistance to, for example, fungi, insects, nematodes and diseases by targeted secretion or accumulation of certain metabolites or proteins in the flower. Examples which may be mentioned are glucosinolates (nematode defense), chitinases or glucanases and other enzymes which destroy the cell wall of parasites, ribosome-inactivating proteins (RIPs) and other proteins of the plant resistance and stress reaction, such as in the case of injury or microbial infestation of plants or chemically by, for example, salicylic acid,
Jasmonsäure oder Ethylen induziert werden, Lysozyme aus nicht-pflanzlichen Quellen wie zum Beispiel T4 Lysozym oder Lysozm aus verschiedenenen Säugern, insektizide Proteine wie Bacillus thuringiensis Endotoxin, a-Amylaseinhibitor oder Proteaseinhibitoren (cowpea Trypsininhibitor), Glucanasen, Lektine (z.B. Phytohemagglutinin, Schneeglöckchenlectin, Weizenkeimagglutinin), RNAsen oder Ribozyme. Besonders bevorzugt sind Nukleinsäuren, die für die chit42 En- dochitinase aus Trichoderma harzianum (GenBank Acc.-No.: S78423) oder für das N-hydroxylierende, multifunktionelle Cytochrom P-450 (CYP79) aus Sorghum bicolor (GenBank Acc.-No.: U32624) oder funktionelle Äquivalente derselben ko- dieren.
4. Erreichen einer Insektenabwehr oder -anlockung zum Beispiel durch erhöhteJasmonic acid or ethylene, lysozymes from non-plant sources such as T4 lysozyme or lysozyme from various mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, α-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. No .: S78423) or for the N-hydroxylating, multifunctional cytochrome P-450 (CYP79) from sorghum bicolor (GenBank Acc.No. : U32624) or functional equivalents thereof. 4. Achieving insect repelling or attracting, for example, through increased
Freisetzung flüchtiger Duft- oder Botenstoffe durch zum Beispiel Enzyme der Ter- penbiosynthese.Release of volatile fragrances or messenger substances by, for example, enzymes of the terpene biosynthesis.
5. Erreichen einer Speicherfähigkeit in Blütengeweben, die normalerweise keine Speicherproteine oder -lipide enthalten mit dem Ziel, den Ertrag an diesen Substanzen zu erhöhen, z.B. durch Expression einer Acetyl-CoA-Carboxylase oder von Enzymen zur Veresterung von Metaboliten. Bevorzugt sind Nukleinsäuren, die für die Acetyl-CoA Carboxylase (Accase) aus Medicago sativa (GenBank Acc.-No.: L25042) oder funktioneile Äquivalente derselben kodieren.5. achieving a storage capacity in flower tissues which normally do not contain storage proteins or lipids with the aim of increasing the yield of these substances, e.g. by expression of an acetyl-CoA carboxylase or of enzymes for the esterification of metabolites. Preference is given to nucleic acids which encode the Medicago sativa acetyl-CoA carboxylase (Accase) (GenBank Acc. No .: L25042) or functional equivalents thereof.
6. Expression von Transportproteinen, die die Aufnahme von Metaboliten, Nährstoffen oder Wasser in die Blüte verbessern und so das Blütenwachstum, die Meta- bolitenzusammensetzung oder den Ertrag optimieren, zum Beispiel durch Ex- pression eines Aminosäuretransporters, der die Aufnahme von Aminosäuren beschleunigt, oder eines Monosaccharid-Transporters, der die Aufnahme von Zuk- kern fördert. Bevorzugt sind Nukleinsäuren, die für den kationische Aminosäure- Transporter aus Arabidopsis thaliana (GenBank Acc.-No.: X92657) oder für den Monosaccharid-Transporter aus Arabidopsis thaliana (Gen-Bank Acc. No.: AJ002399) oder funktionelle Äquivalente deselben kodieren.6. Expression of 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. Preference is given to 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.
7. Expression von Genen, die eine Akkumulation von Feinchemikalien, wie von To- copherolen, Tocotrienolen, Phenylpropanoiden, Isoprenoiden oder Carotiniden, in der Blüte bewirken. Beispielhaft seien die Deoxyxylulose-5-phosphatsynthasen, Phytoensynthasen, Lycopin-b-cyklasen und die b-Carotinketolasen genannt. Bevorzugt sind Nukleinsäuren, die für die Haematoccus pluvialis NIES-144 (Acc. No. D45881) Ketolase oder funktionelle Äquivalente derselben kodieren.7. Expression of 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.
8. Modifikation der Wachsesterbildung oder der Zusammensetzung der eingelager- ten Oligosaccharide zur Verbesserung des Schutzes gegen Umwelteinflüsse oder zur Verbesserung der Verdaubarkeit beim Einsatz in Futter- oder Nahrungsmitteln. Beispielhaft sein die Überexpression der Endoxyloglucantransferase genannt. Bevorzug sind Nukleinsäuren, die für die Endo-xyloglucantransferase (EXGT-AI) aus Arabidopsis thaliana (Gen-Bank Acc.-No. :AF163819} oder funktio- nelle Äquivalente derselben kodieren.8. modification of the wax ester formation or the composition of the stored oligosaccharides to improve the protection against environmental influences or to improve the digestibility when used in feed or food. As an example, the overexpression of the endoxyloglucan transferase may be mentioned. Preferred are nucleic acids which code for the endo-xyloglucan transferase (EXGT-AI) from Arabidopsis thaliana (GenBank Acc. No: AF163819) or functional equivalents thereof.
9. Expression von Genen, DNA Bindeproteinen, dsRNA und antisense Konstruktionen, zur Veränderung der Blütenmorphologie, des Blühzeitpunktes und der Blü- tenseneszenz sowie des Blütenmetabolismus. Bevorzugt sind Konstruktionen, die die Anzahl der Petalen erhöhen z.B. durch Herunterregulation von AGAMOUS und dessen homologen Genen (Yanofsky MF et al. (1990) Nature 346:35-39) den Blühzeitpunkt verfrühen z.B. durch Herunterregulation von FLOWERING LOCUS
C (FLC) (Tadege M et al. (2001) Plant J 28(5):545-53) oder verspäten z.B. durch Überexpression von FLC und die Seneszenz verzögern z.B. durch Vermittlung einer blütenspezifischen Ethyleninsensitivität.9. Expression of Genes, DNA Binding Proteins, dsRNA and Antisense Constructions, for the Modification of the Flower Morphology, the Flowering Time and the Bloom Elsecence as well as the Flower Metabolism. Preference is given to constructions which increase the number of petals, for example by down-regulation of AGAMOUS and its homologous genes (Yanofsky MF et al. (1990) Nature 346: 35-39), pruning the flowering time, for example by down-regulation of FLOWERING LOCUS C (FLC) (Tadege M et al. (2001) Plant J 28 (5): 545-53) or delayed, for example, by overexpression of FLC and retard senescence, eg, by mediating flower-specific ethylene insensitivity.
10. Erzeugung von sterilen Pflanzen durch Verhinderung der Befruchtung und/oder der Keimung mit Hilfe der Expression eines geeigneten Inhibitors zum Beispiel eines Toxins in Blüten.10. Production of sterile plants by preventing fertilization and / or germination by expression of a suitable inhibitor, for example, of a toxin in flowers.
11. Produktion von Nutraceuticals wie zum Beispiel11. Production of nutraceuticals such as
a) Carotinoide und/oder Phenylpropanoide z.B. durch Optimierung der blüteneigenen Stoffwechselwege z.B. durch Expression von Enzymen und Regulatoren der Isoprenoidbiosynthese. Bevorzugt sind Nukleinsäuren, die für die Chalconsynthase aus Arabidopsis thaliana (GenBank Acc.-No.: M20308), die 6-4 Photolyase aus Arabidopsis thaliana (GenBank Acc.No.:BAB00748) oder den Blaulicht-Photorezeptor / Photolyase Homolog (PHHI) aus Arabidopsis thaliana (GenBank Acc.-No.: U62549) oder funktionelle Äquivalente derselben kodieren. Ebenso bevorzugt sind Nukleinsäuren, die für Enzyme und Regulatoren der Isoprenoidbiosynthese wie die Deoxyxylulose-5- phosphatsynthasen und der Carotinoidbiosynthese wie die Phytoensyntha- sen, Lycopincyclasen und Ketolasen wie von Tocopherolen, Tocotrienolen, Phenylpropanoiden, Isoprenoiden oder Carotiniden, in der Blüte bewirken. Beispielhaft seien die Deoxyxylulose-5-phosphatsynthasen, Phytoensyntha- sen, Lycopincyclasen und die Carotinketolasen genannt. Besonders devor- zugt sind Nukleinsäuren, die für die Haematoccus pluvialis, NIES-144 (Acc.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. Likewise preferred are 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. Examples which may be mentioned are the deoxyxylulose-5-phosphate synthases, phytoene synthases, lycopene cyclases and the carotene toetolases. Particular preference is given to nucleic acids which are useful for Haematoccus pluvialis, NIES-144 (Acc.
No. D45881) Ketolase oder funktionelle Äquivalente kodieren.No. D45881) Encoding ketolase or functional equivalents.
b) Polyungesättigte Fettsäuren wie beispielsweise Arachidonsäure oder EP (Eicosapentaensäure) oder DHA (Docosahexaensäure) durch Expression von Fettsäureelongasen und/oder - desaturasen oder Produktion von Proteinen mit verbessertem Nahrungswert wie zum Beispiel mit einem hohen Anteil an essentiellen Aminosäuren (z.B. das methioninreiche 2S Albumingens der Brasilnuss). Bevorzugt sind Nukleinsäuren, die für das methioninreiche 2S-Albumin aus Bertholletia excelsa (GenBank Acc.-No. :AB044391), die D6- Acyllipiddesaturase aus Physcomitrella patens (GenBank Acc.-No.:b) Polyunsaturated fatty acids such as arachidonic acid or EP (eicosapentaenoic acid) or DHA (docosahexaenoic acid) by expression of fatty acid elongases and / or desaturases or production of proteins with improved nutritional value, for example with a high proportion of essential amino acids (eg the methionine - rich 2S albumin gene of Brazil nut). Preference is given to nucleic acids which are suitable for the methionine-rich 2S albumin from Bertholletia excelsa (GenBank Acc. No: AB044391), the D6 acyl lipid desaturase from Physcomitrella patens (GenBank Acc.
AJ222980; Girke et al. (1998) Plant J 15:39-48), die D6-Desaturase aus Mor- tierella alpina (Sakura-dani et al 1999 Gene 238:445-453), die D5- Desaturase aus Caenorhabditis elegans (Michaelson et al. (1998) FEBS Letters 439:215-218), die D5-Fettsäuredesaturase (des-5) aus Caenorhabditis elegans (GenBank Acc.-No.: AF078796), die D5-Desaturase aus Mortierella alpina (Michaelson et al. J Biol Chem 273:19055-19059), die D6-Elongase aus Caenorhabditis elegans (Beaudoin et al. (2000) Proc Natl. Acad. Sei.
97:6421-6426), die A6-Elongase aus Physcomitrella patens (Zank et al. (2000,) Biochemical Society Transactions 28:654-657) oder funktionelle Äquivalente derselben kodieren.AJ222980; Girke et al. (1998) Plant J 15: 39-48), the Moretonella alpina D6 desaturase (Sakura-dani et al 1999 Gene 238: 445-453), the D5 desaturase from Caenorhabditis elegans (Michaelson et al FEBS Letters 439: 215-218), the D5-fatty acid desaturase (des-5) from Caenorhabditis elegans (GenBank Acc. No .: AF078796), the Mortierella alpina D5 desaturase (Michaelson et al., J. Biol. Chem. 19055-19059), the D6 elongase from Caenorhabditis elegans (Beaudoin et al. (2000) Proc Natl. Acad. 97: 6421-6426) encoding A6 elongase from Physcomitrella patens (Zank et al., (2000) Biochemical Society Transactions 28: 654-657) or functional equivalents thereof.
12. Produktion von Pharmazeutika, wie zum Beispiel Antikörpern, Vakzinen, Hormonen und/oder Antibiotika wie z.B. beschrieben bei Hood EE & Jilka JM (1999) Curr Opin Biotechnol 10(4):382-6; Ma JK & Vine ND (1999) CurrTop Microbiol Immunol 236:275-92.12. Production of pharmaceuticals, such as antibodies, vaccines, hormones and / or antibiotics, e.g. described by Hood EE & Jilka JM (1999) Curr Opin Biotechnol 10 (4): 382-6; Ma JK & Vine ND (1999) CurrTop Microbiol Immunol 236: 275-92.
Weitere Beispiele für vorteilhafte Gene sind zum Beispiel genannt bei Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot. 51 Spec No:487-96.Further examples of advantageous genes are mentioned, for example, in Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot. 51 Spec No: 487-96.
Ein weiterer Gegenstand der Erfindung betrifft die Verwendung der oben beschriebenen erfindungsgemässen, transgenen Organismen und der von ihnen abgeleitete ZeI- len, Zellkulturen, Teile - wie zum Beispiel bei transgenen pflanzlichen Organismen Wurzeln, Blätter etc.- , und transgenes Vermehrungsgut wie Saaten oder Früchte, zur Herstellung von Nahrungs- oder Futtermitteln, Pharmazeutika oder Feinchemikalien.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.
Bevorzugt ist ferner ein Verfahren zur rekombinanten Herstellung von Pharmazeutika oder Feinchemikalien in Wirtsorganismen, wobei ein Wirtsorganismus mit einer der oben beschriebenen Expressionskassetten transformiert wird und diese Expressionskassette ein oder mehrere Strukturgene enthält, die für die gewünschte Feinchemikalie kodieren oder deren Biosynthese katalysieren, der transformierte Wirtsorganismus gezüchtet wird und die gewünschte Feinchemikalie aus dem Züchtungsmedium isoliert wird. Dieses Verfahren ist für Feinchemikalien wie Enzyme, Vitamine, Aminosäuren, Zucker, Fettsäuren, natürliche und synthetische Geschmacks-, Aroma- und Farbstoffe breit anwendbar. Besonders bevorzugt ist die Produktion von Tocopherolen und Toco- trienolen sowie Carotinoiden wie beispielsweise Astaxanthin. Die Züchtung der transformierten Wirtsorganismen sowie die Isolierung aus den Wirtsorganismen bzw. aus dem Züchtungsmedium erfolgt mit dem Fachmann bekannten Verfahren. Die Produktion von Pharmazeutika, wie zum Beispiel Antikörpern oder Vakkzinen ist beschrieben bei Hood EE & Jilka JM (1999) Curr Opin Biotechnol 10 (4)382-6; Ma JK & Vine ND (1999) Curr Top Microbiol Immunol 236:275-92.Also preferred is a process for the recombinant production of pharmaceuticals or fine chemicals in host organisms, wherein a host organism is transformed with one of the expression cassettes described above, and that expression cassette contains one or more structural genes encoding the desired fine chemical or catalyzing its biosynthesis, the transformed host organism and the desired fine chemical is isolated from the culture medium. This process is widely applicable to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavorings, flavorings and dyes. Particularly preferred is the production of tocopherols and tocotrienols as well as carotenoids such as astaxanthin. 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. The production of pharmaceuticals, such as antibodies or vaccines, is described in Hood EE & Jilka JM (1999) Curr Opin Biotechnol. 10 (4) 382-6; Ma JK & Vine ND (1999) Curr Top Microbiol Immunol 236: 275-92.
Sequenzen:sequences:
1. SEQ ID NO: 1 2554bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g333701. SEQ ID NO: 1 2554bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g33370
2. SEQ ID NO: 2 Funktionell äquivalentes Fragment (1541 bp) von Promotor und2. SEQ ID NO: 2 Functionally equivalent fragment (1541 bp) of promoter and
5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g33370
3. SEQ ID NO: 3 Funktionell äquivalentes Fragment (668 bp) von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g333705'-untranslated region of the Arabidopsis thaliana gene locus At5g33370 3. SEQ ID NO: 3 Functionally equivalent fragment (668 bp) of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g33370
4. SEQ ID NO: 4 2103bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g224304. SEQ ID NO: 4 2103bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g22430
5. SEQ ID NO: 5 Funktionell äquivalentes Fragment (1376 bp) von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus5. SEQ ID NO: 5 Functionally equivalent fragment (1376 bp) of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus
At5g22430At5g22430
6. SEQ ID NO: 6 Funktionell äquivalentes Fragment (746 bp) von Promotor und6. SEQ ID NO: 6 Functionally equivalent fragment (746 bp) of promoter and
5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g224305'-untranslated region of the Arabidopsis thaliana gene locus At5g22430
7. SEQ ID NO: 7 2945bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At1g266307. SEQ ID NO: 7 2945 bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At1g26630
8. SEQ ID NO: 8 Funktionell äquivalentes Fragment (1628 bp) von Promotor und8. SEQ ID NO: 8 Functionally equivalent fragment (1628 bp) of promoter and
5'-untranslatierter Region des Arabidopsis thaliana Genlocus At1g266305'-untranslated region of the Arabidopsis thaliana gene locus At1g26630
9. SEQ ID NO: 9 Funktionell äquivalentes Fragment (587 bp) von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus9. SEQ ID NO: 9 Functionally equivalent fragment (587 bp) of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus
At1g26630At1g26630
10. SEQ ID NO: 10 2572bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At4g3510010. SEQ ID NO: 10 2572bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At4g35100
11. SEQ ID NO: 11 2421 bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At3g0429011. SEQ ID NO: 11 2421 bp fragment of promoter and 5'-untranslated region of the Arabidopsis thaliana gene locus At3g04290
12. SEQ ID NO: 12 2345bp Fragment von Promotor und 5'-untranslatierter Region des Arabidopsis thaliana Genlocus At5g4611012. SEQ ID NO: 12 2345 bp Fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana gene locus At5g46110
13. SEQ ID NO: 13 Oligonukleotidprimer M1as13. SEQ ID NO: 13 oligonucleotide primer M1as
14. SEQ ID NO: 14 Oligonukleotidprimer M1s14. SEQ ID NO: 14 oligonucleotide primer M1s
15. SEQ ID NO: 15 Oligonukleotidprimer Miss
16. SEQ ID NO: 16 Oligonukleotidprimer M1svl15. SEQ ID NO: 15 oligonucleotide primer Miss 16. SEQ ID NO: 16 oligonucleotide primer M1svl
17. SEQ ID NO: 17 Oligonukleotidprimer M2as17. SEQ ID NO: 17 oligonucleotide primer M2as
18. SEQ ID NO: 18 Oligonukleotidprimer M2s18. SEQ ID NO: 18 oligonucleotide primer M2s
19. SEQ ID NO: 19 Oligonukleotidprimer M2ss19. SEQ ID NO: 19 oligonucleotide primer M2ss
20. SEQ ID NO: 20 Oligonukleotidprimer M2svl20. SEQ ID NO: 20 oligonucleotide primer M2svl
21. SEQ ID NO: 21 Oligonukleotidprimer M3as21. SEQ ID NO: 21 oligonucleotide primer M3as
22. SEQ ID NO: 22 Oligonukleotidprimer M3s22. SEQ ID NO: 22 oligonucleotide primer M3s
23. SEQ ID NO: 23 Oligonukleotidprimer M3ss23. SEQ ID NO: 23 oligonucleotide primer M3ss
24. SEQ ID NO: 24 Oligonukleotidprimer M3svl24. SEQ ID NO: 24 oligonucleotide primer M3svl
25. SEQ ID NO: 25 Oligonukleotidprimer M4as25. SEQ ID NO: 25 oligonucleotide primer M4as
26. SEQ ID NO: 26 Oligonukleotidprimer M4s26. SEQ ID NO: 26 oligonucleotide primer M4s
27. SEQ ID NO: 27 Oligonukleotidprimer M5as27. SEQ ID NO: 27 oligonucleotide primer M5as
28. SEQ ID NO: 28 Oligonukleotidprimer M5s28. SEQ ID NO: 28 oligonucleotide primer M5s
29. SEQ ID NO: 29 Oligonukleotidprimer M6as29. SEQ ID NO: 29 oligonucleotide primer M6as
30. SEQ ID NO: 30 Oligonukleotidprimer M6s30. SEQ ID NO: 30 oligonucleotide primer M6s
BeispieleExamples
Allgemeine Methoden:General methods:
Die chemische Synthese von Oligonukleotiden kann beispielsweise, in bekannter Weise, nach der Phosphoamiditmethode (Voet & Voet (1995), 2. Auflage, Wiley Press New York, Seite 896-897) erfolgen. Die im Rahmen der vorliegenden Erfindung durchgeführten Klonierungsschritte wie z.B. Restriktionsspaltungen, Agarosegelelektrophorese, Reinigung von DNA-Fragmenten, Transfer von Nukleinsäuren auf Nitrozellulose und Nylonmembranen, Verknüpfen von DNA-Fragmenten, Transformation von E. coli Zellen, Anzucht von Bakterien, Vermehrung von Phagen und Sequenzanalyse rekombi- nanter DNA werden wie bei Sambrook et al. (1989) CoId Spring Harbor Laboratory
Press; ISBN 0-87969-309-6 beschrieben durchgeführt. Die Sequenzierung rekombi- nanter DNA-Moleküle erfolgt mit einem Laserfluoreszenz-DNA-Sequenzierer der Firma ABI nach der Methode von Sanger (Sanger et al.(1977) Pro Natl Acad Sei USA 74:5463-5467).The chemical synthesis of 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).
Zur Herstellung transgener Arabidopsis Pflanzen wird Agrobakterium tumefaciens (Stamm C58C1 pMP90) mit verschiedenen Promotor-GUS Vektorkonstrukten transformiert. Die Agrobakterienstämme werden anschliessend zur Herstellung transgener Pflanzen verwendet. Dazu wird eine einzelne transformierte Agrobakterium Kolonie in einer 4 ml Kultur (Medium: YEB-Medium mit 50 μg/ml Kanamycin und 25 μg/ml Rifampicin über Nacht bei 28°C inkubiert. Mit dieser Kultur wird anschliessend eine 400 ml Kultur in demselben Medium angeimpft, über Nacht inkubiert (28 °C, 220 U/min) und abzentrifugiert (GSA-Rotor, 8.000 U/min, 20 min). Das Pellet wird in Infiltrationsmedium (1/2 MS-Medium; 0,5 g/l MES, pH 5,8; 50 g/l Saccharose) resuspendiert. Die Suspen- sion wird in eine Pflanzenbox (Duchefa) eingebracht und 100 ml SILVET L-77 (mit Po- lyalkylenoxid modifiziertes Heptamethyltrisiloxan; Osi Specialties Inc., Cat. P030196) wurde zu einer Endkonzentration von 0.02% hinzugegeben. Die Pflanzenbox mit 8 bis 12 Pflanzen wird in einem Exikator für 10 bis 15 Minuten einem Vakuum mit anschlie- ssender spontaner Belüftung ausgesetzt. Dies wird 2 bis 3 Mal wiederholt. Hernach werden alle Pflanzen in Pflanztöpfe mit Feuchterde gepflanzt und unter Langtagbedingungen (16 h Beleuchtung) gezüchtet (Tagestemperatur 22 bis 24°C, Nachtemperatur 19°C; 65 % relative Luftfeuchte). Nach 6 Wochen werden die Samen geerntet.To produce transgenic Arabidopsis plants, 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 μg / ml kanamycin and 25 μg / 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.
Beispiel 1 : Wachstumsbedingungen der Pflanzen für gewebsspezifische RT-PCR AnalyseExample 1: Plant growth conditions for tissue-specific RT-PCR analysis
Um 4 bzw. 7 Tage alte Keimlinge zu erhalten, werden jeweils ungefähr 400 Samen (Arabidopsis thaliana ökotyp Columbia) oberflächig mit einer 80%igen Ethanollösung für 2 Minuten sterilisiert, mit einer Natriumhypochloritlösung (0.5 % v/v) 5 Minuten be- handelt, dreimal mit destilliertem Wasser gewaschen und bei 4°C für 4 Tage inkubiert, um eine gleichmässige Keimung sicherzustellen. Anschliessend werden die Samen auf Petrischalen mit MS Medium (Sigma M5519) unter Zusatz von 1% Saccharose, 0.5 g/l MES (Sigma M8652), 0.8 % Difco-BactoAgar (Difco 0140-01), pH 5.7 inkubiert. Die Sämlinge werden in einem 16-stündigen Licht / 8-stündigen Dunkelheitszyklus (Philips 58W/33 Weisslichtlampen) bei 22°C gezüchtet und nach 4 Tagen nach Beginn der Keimphase geerntet.To obtain 4 or 7 day old seedlings, about 400 seeds (Arabidopsis thaliana ecotype Columbia) are surface sterilized with an 80% ethanol solution for 2 minutes, treated with a sodium hypochlorite solution (0.5% v / v) for 5 minutes, washed three times with distilled water and incubated at 4 ° C for 4 days to ensure even germination. Subsequently, the seeds are incubated on Petri dishes with MS medium (Sigma M5519) with addition of 1% sucrose, 0.5 g / l MES (Sigma M8652), 0.8% Difco-BactoAgar (Difco 0140-01), 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 and harvested after 4 days from the start of the germination phase.
Für die Gewinnung von Wurzeln werden 100 Samen wie oben beschrieben sterilisiert, bei 4°C 4 Tage inkubiert und dann in 250ml Flaschen mit MS Medium (Sigma M5519) unter Zusatz von weiteren 3 % Saccharose und 0.5 g/l MES (Sigma M8652), pH 5.7 gezüchtet. Die Sämlinge werden in einem 16-stündigen Licht- / 8-stündigen Dunkelheitszyklus (Philips 58W/33 Weisslichtlampen) bei 22°C, 120 U/min gezüchtet und
nach 3 Wochen geerntet. Für alle anderen verwendeten pflanzlichen Organe werden die Samen auf Einheitserde (Typ VM, Manna-Italia, Via S. Giacomo 42, 39050 San Giacomo/ Laives, Bolzano, Italien) ausgesäht, 4 Tage bei 4°C inkubiert um eine gleichmäßige Keimung zu gewährleisten und dann in einem 16-stündigen Licht- / 8- stündigen Dunkelheitszyklus (OSRAM Lumi-lux Daylight 36W/12 Leuchtstoffröhren) bei 22°C gezüchtet. Junge Rosettenblätter werden im 8-Blattstadium (nach 3 Wochen) geerntet, reife Rosetten blätter werden nach 8-Wochen kurz vor der Stengelbildung geerntet. Blütenstände (Apices) der ausschießenden Stengel werden kurz nach dem Ausschießen geerntet. Stengel, Stengelblätter und Blütenknospen werden in der Ent- wicklungsstufe 12 (Bowmann J (ed.), Arabidopsis, Atlas of Morphology, Springer New York, 1995) vor der Staubblattentwicklung geerntet. Geöffnete Blüten werden in Stadium 14 sofort nach der Staubblattentwicklung geerntet. Welkende Blüten werden in Stadium 15 bis 16 geerntet. Die verwendeten grünen und gelben Schoten hatten eine Länge von 10 bis 13 mm.For rooting, 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. For all other plant organs used, 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. to ensure uniform germination and then grown in a 16-hour light / 8-hour dark cycle (OSRAM Lumi-lux Daylight 36W / 12 fluorescent tubes) at 22 ° C. Young rosette leaves are harvested at the 8-leaf stage (after 3 weeks), mature rosette leaves are harvested after 8 weeks just before stalking. Inflorescences (apices) of the shooting stems are harvested shortly after imposition. Stems, stem leaves and flower buds are harvested at development stage 12 (Bowman J (ed.), Arabidopsis, Atlas of Morphology, Springer New York, 1995) prior to stamen development. Open flowers are harvested at stage 14 immediately after stamen development. Withering flowers are harvested in stages 15 to 16. The green and yellow pods used had a length of 10 to 13 mm.
Beispiel 2: Nachweis der gewebespezifischen ExpressionExample 2: Detection of tissue-specific expression
Um die Eigenschaften des Promotors zu bestimmen und die essentiellen Elemente desselben, die seine Gewebespezifität ausmachen, zu identifizieren, ist es erforderlich, den Promotor selbst und verschiedene Fragmente desselben vor ein sogenanntes Reportergen zu setzen, das eine Bestimmung der Expressionsaktivität ermöglicht. Beispielhaft sei die bakterielle ß-Glucuronidase genannt (Jefferson et al. (1987) EMBO J 6:3901-3907). Die ß-Glucuronidase Aktivität kann in planta mittels eines chromogenen Substrates wie 5-Bromo-4-Chloro-3-lndolyl-ß-D-Glucuronsäure im Rahmen einer Akti- vitätsfärbung bestimmt werden (Jefferson et al. (1987) Plant Mol Biol Rep 5:387-405). Für die Untersuchungen der Gewebespezifität wird das pflanzliche Gewebe geschnitten, eingebettet, gefärbt und analysiert wie beschrieben (z.B. Bäumlein H et al. (1991) Mol Gen Genet 225:121-128).In order to determine the properties of the promoter and to identify the essential elements thereof that make up its tissue specificity, it is necessary to place the promoter itself and various fragments thereof in front of a so-called reporter gene, which allows a determination of the expression activity. By way of example, bacterial β-glucuronidase may be mentioned (Jefferson et al. (1987) EMBO J 6: 3901-3907). The β-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). For tissue specificity studies, the plant tissue is cut, embedded, stained, and analyzed as described (e.g., Baumlein et al., (1991) Mol Gen Genet 225: 121-128).
Für die quantitative Aktivitätsbestimmung der ß-Glucuronidase wird als Substrat MUG (Methylumbelliferylglucuronid) verwendet, das in MU (Methylumbelliferon) und Glucu- ronsäure gespalten wird. Unter alkalischen Bedingungen kann diese Spaltung quantitativ fluorometrisch verfolgt werden (Anregung bei 365 nm, Messung der Emission bei 455 nm; SpectroFluorimeter Thermo Life Sciences Fluoroscan) wie beschrieben (Bu- stos MM et al. (1989) Plant Gell 1 :839-853).For the quantitative activity determination of β-glucuronidase, 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). ,
Beispiel 3: Klonierung der PromotorenExample 3: Cloning of the promoters
Um die vollständigen Promotoren gemäß 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 und SEQ ID NO: 12 zu isolieren, wird ge-
nomische DNA aus Arabidopsis thaliana (ökotyp Landsberg erecta) extrahiert wie beschrieben (Galbiati M et al. Funct. Integr. Genomics 2000, 20 1 :25-34). Die isolierte DNA wird als Matrizen-DNA in einer PCR unter Verwendung folgender Oligonukleotid Primer-Kombinationen und annealing Temperaturen eingesetzt:In order to obtain the complete promoters according to 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:
Die Amplifikation wird wie folgt durchgeführt:The amplification is carried out as follows:
80 ng genomische DNA 1X Expand™ Long Template PCR Puffer80 ng genomic DNA 1X Expand ™ Long Template PCR buffer
2,5 rtiM MgCI2, je 350 μM von dATP, dCTP, dGTP und dTTP je 300 nM eines jeden Primers2.5 μM MgCl 2, 350 μM each of dATP, dCTP, dGTP and dTTP each 300 nM of each primer
2,5 Units Expand™ Long Template Polymerase (Roche Diagnostics). in einem Endvolumen von 25 μl2.5 Units Expand ™ Long Template Polymerase (Roche Diagnostics). in a final volume of 25 μl
Folgendes Temperaturprogramm wird verwendet (PTC-100TM Modell QW; MJ Research, Inc., Watertown, Massachussetts):The following temperature program is used (PTC-100 ™ Model QW; MJ Research, Inc., Watertown, Massachusetts):
1 Zyklus mit 120 sec bei 94°C1 cycle with 120 sec at 94 ° C
35 Zyklen mit 94°C für 10 sec, für 30 sec die in Tabelle 1 angegebene Temperatur und 68°C für 3min. 1 Zyklus mit 68°C für 30 min 4535 cycles at 94 ° C for 10 sec, for 30 sec the temperature given in Table 1 and 68 ° C for 3 min. 1 cycle at 68 ° C for 30 min 45
Für die Amplifikation der Fragmente wurden Oligonukleotide als Primer verwendet, die an ihren 5Λ-Termini Phosphatreste tragen. Dadurch ist eine direkte Klonierung der Promotoren in den mit der Restriktionendonuklease Smal geöffneten Vektor pS0301
(Fig.1) möglich. Der Vektor pS0301 enthält 3'der Smal Schnittstelle die kodierende Sequenz des GUS-Reportergens. Durch Klonierung der Promotorfragmente gemäß 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 und SEQ ID NO: 12 entstanden Genfusionen aus den Promotorfragmenten und der ß- Glucuronidase (GUS).For the amplification of the fragments oligonucleotides were used as primers, which carry at their 5 Λ -Termini phosphate residues. This is a direct cloning of the promoters in the open with the restriction endonuclease Smal vector pS0301 (Fig.1) possible. The vector pS0301 contains 3 'of the SmaI site the coding sequence of the GUS reporter gene. By cloning the promoter fragments of 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 β-glucuronidase (GUS).
Nach der Agrobakterium tumefaciens vermittelten Transformation dieser Konstrukte in das Genom von Arabidopsis thaliana kann die Expression des GUS Gens mittels hi- stochemischer Färbemethoden visualisert werden.After the Agrobacterium tumefaciens mediated transformation of these constructs into the genome of Arabidopsis thaliana, the expression of the GUS gene can be visualized by means of histochemical staining methods.
Beispiel 4: TAIL-PCRExample 4: TAIL-PCR
Die "TAIL-PCR" wird entsprechend einem adaptrierten Protokoll der Methode von Liu et al. (1995) Plant J 8(3):457-463 und Tsugeki et al. (1996) Plant J 10(3):479-489 durchgeführt (vgl. Fig. 9). Für eine erste PCR-Reaktion wird folgender Mastermixes (Angaben pro Reaktionsansatz) eingesetztThe "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). For a first PCR reaction, the following master mix (data per reaction mixture) is used
11 μl steriles H20 (bidestilliert) 2 μl Primer-Stocklösung des spezifischen Primers 1 (5mM) 3 μl AD2 Primer-Stocklösung (2OmM) 2 μl 10x-PCR-Puffer 2 μl 10xdNTP 0,2 μl Taq Polymerase11 μl sterile H 2 O (bidistilled) 2 μl primer stock solution of specific primer 1 (5 mM) 3 μl AD 2 primer stock solution (2 mM) 2 μl 10 × PCR buffer 2 μl 10 × dNTP 0.2 μl Taq polymerase
19 μl dieses Mastermixes werden in einem PCR-Gefäß zu 1 μl einer Präparation genomischer DNA des jeweiligen Zielorganismus (Präparation gemäß Galbiati M et al. (2000) Funct Integr Genozides 20(1):25-34)) hinzupipettiert und durch Pipettieren gut gemischt. Die primäre PCR-Reaktion wird unter folgenden Bedingungen durchgeführt:19 μl of this master mix are pipetted into 1 μl of a preparation of genomic DNA of the respective target organism (preparation according to Galbiati M et al. (2000) Funct Integr Genozides 20 (1): 25-34)) and mixed well by pipetting , The primary PCR reaction is carried out under the following conditions:
- 94°C für 1 min- 94 ° C for 1 min
- Vier Zyklen mit 94°C für 10s, 62°C für 1 min und 72°C für 150s- Four cycles at 94 ° C for 10s, 62 ° C for 1 min and 72 ° C for 150s
- 94°C für 10s, 25°C für 3 min, 0,2°C/s bis 72°C und 72°C für 150s- 94 ° C for 10 s, 25 ° C for 3 min, 0.2 ° C / s to 72 ° C and 72 ° C for 150 s
- Vierzehn Zyklen mit 94°C für 10s, 69°C für 1 min, 72°C für 150s, 94°C für 10s, 68°C für 1 min, 72°C für 150s, 94°C für 10s, 44°C für 1 min und 72°C für 150s- Fourteen cycles at 94 ° C for 10s, 69 ° C for 1 min, 72 ° C for 150s, 94 ° C for 10s, 68 ° C for 1 min, 72 ° C for 150s, 94 ° C for 10s, 44 ° C for 1 min and 72 ° C for 150s
- 72°C für 5 min, dann 4°C bis zur Weiterverwendung.
Das Produkt der PCR-Reaktion wird 1 :50 verdünnt und je 1 μl jeder verdünnten Probe wird für eine zweite PCR-Reaktion (sekundäre PCR) verwendet. Dazu wird folgender Mastermix (Angaben pro Reaktionsansatz) eingesetzt:- 72 ° C for 5 min, then 4 ° C until further use. The product of the PCR reaction is diluted 1:50 and 1 μl of each diluted sample is used for a second PCR reaction (secondary PCR). For this purpose, the following master mix (data per reaction mixture) is used:
12 μl steriles H2O (bidestilliert)12 μl sterile H2O (bidistilled)
2 μl 10x-PCR-Puffer (1,5 mM MgCI2)2 μl of 10x PCR buffer (1.5 mM MgCl 2)
2 μl 10xdNTP2 μl 10xdNTP
2 μl Primer-Stocklösung des spezifischen Primers 2 (5 mM)2 μl primer stock solution of specific primer 2 (5 mM)
2 μl AD2 Primer-Stocklösung 0,2μl Taq Polymerase2 μl AD2 primer stock solution 0.2 μl Taq polymerase
Je 20,2 μl des zweiten Mastermix werden zu je 1 μl des 1 :50 verdünnten primären PCR-Produktes gegeben und die sekundäre PCR wird unter folgenden Bedingungen durchgeführt:20.2 μl each of the second master mix are added to 1 μl each of the 1:50 diluted primary PCR product and the secondary PCR is carried out under the following conditions:
- 11 Zyklen mit 94°C für 10s, 64°C für 1 min, 72°C für 150s, 94°C für 10s, 64°C für 1 min, 72°C für 150s, 94°C für 10s, 44°C für 1 min, 72°C für 150s,- 11 cycles at 94 ° C for 10s, 64 ° C for 1 min, 72 ° C for 150s, 94 ° C for 10s, 64 ° C for 1 min, 72 ° C for 150s, 94 ° C for 10s, 44 ° C for 1 min, 72 ° C for 150s,
72°C für 5min, dann 4°C bis zur Weiterverwendung.72 ° C for 5min, then 4 ° C until further use.
Das PCR-Produkt der vorhergehenden Reaktion wird 1 :10 verdünnt und je 1 μl jeder verdünnten Probe wird für eine dritte PCR-Reaktion (tertiäre PCR) verwendet. Dazu wird folgender Mastermix (Angaben pro Reaktionsansatz) eingesetzt:The PCR product of the previous reaction is diluted 1:10 and 1 μl of each diluted sample is used for a third PCR reaction (tertiary PCR). For this purpose, the following master mix (data per reaction mixture) is used:
18 μl steriles H2O (bidestilliert)18 μl sterile H2O (bidistilled)
3 μl 10x-PCR-Puffer (1,5 mM MgCI2) 3 μl 10xdNTP3 μl 10x PCR Buffer (1.5 mM MgCl 2) 3 μl 10xdNTP
3 μl Primer-Stocklösung des spezifischen Primers 3 (5mM) 3 μl AD2 Primer-Stocklösung 0,5 μl Taq Polymerase3 μl primer stock solution of specific primer 3 (5mM) 3 μl AD2 primer stock solution 0.5 μl Taq polymerase
Je 30,3 μl dieses Mastermixes werden zu je 1 μl des 1 :10 verdünnten sekundären PCR-Produktes gegeben und die tertiäre PCR wird unter folgenden Bedingungen durchgeführt:In each case 30.3 μl of this master mix are added to 1 μl each of the 1:10 diluted secondary PCR product and the tertiary PCR is carried out under the following conditions:
- 19 Zyklen mit 94°C für 15s, 44°C für 1 min, 72°C für 150s,- 19 cycles at 94 ° C for 15s, 44 ° C for 1 min, 72 ° C for 150s,
72°C für 5 min, dann 4°C bis zur Weiterverwendung.72 ° C for 5 min, then 4 ° C until further use.
Von den Produkten der PCR 1 , 2 und 3 jeder Probe werden je 5 μl auf einem 2%igen Agarosegel aufgetrennt. Diejenigen PCR-Produkte, die wegen der versetzten spezifischen Primer die erwartete Größenverringerung aufweisen, werden bei Bedarf aus
dem Gel gereinigt und mit dem zuletzt verwendeten Primerpaar erneut amplifiziert und sequenziert.Of the products of PCR 1, 2 and 3 of each sample are each 5 ul separated on a 2% agarose gel. Those PCR products which have the expected size reduction due to the staggered specific primers will turn out as needed the gel was purified and re-amplified with the last used primer pair and sequenced.
Reagenzien:reagents:
Taq-Polymerase 5U/μlTaq polymerase 5U / μl
1Ox PCR-Puffer (1,5 rtiM MgCI2)1 x PCR buffer (1.5 rtiM MgCl 2)
1Ox dNTP-Stocklösung: 2 rtiM1Ox dNTP stock solution: 2 rtiM
Primer:primer:
Degenerierte Zufallsprimer (Stocklösungen 20 μM): 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'Degenerate random primers (stock solutions 20 μM): 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 '
Beispiel 6: Inverse PCR (iPCR) für die Amplifikation Insert-flankierdender DNAExample 6: Inverse PCR (iPCR) for amplification of insert-flanking DNA
Die "iPCR" wird entsprechend einem adaptierten Protokoll der Methode von Long et al.(1993) PNAS 90:10370 durchgeführt:The "iPCR" is performed according to an adapted protocol of the method of Long et al. (1993) PNAS 90: 10370:
1. Restriktion von ca. 2 μg genomischer DNA mit BstYl für ca. 2h bei 37°C in einem Volumen von insgesamt 50 μl.1. Restriction of approx. 2 μg of genomic DNA with BstYI for approx. 2 h at 37 ° C. in a total volume of 50 μl.
2. Ligation von 25 μl des Restriktionsansatzes in einem Gesamtvolumen von 300 μl mit 3U T4-DNA-Ligase bei 15°C über Nacht.2. Ligation of 25 .mu.l of the restriction mixture in a total volume of 300 .mu.l with 3U T4 DNA ligase at 15 ° C overnight.
3. Phenol/Chloroform-Extraktion und anschließende Chloroform Extraktion des Liga- tionsansatzes. Nach Ethanolfällung DNA in 10 μl sterilem H2O (bidestilliert) aufnehmen.3. Phenol / chloroform extraction followed by chloroform extraction of the ligation mixture. After ethanol precipitation, take up DNA in 10 μl of sterile H2O (bidistilled).
4. Für die PCR 2,5μl der DNA-Lösung einsetzen4. Use 2.5μl of the DNA solution for the PCR
Reaktionsansatz:Reaction:
2,5 μl der DNA-Lösung2.5 μl of the DNA solution
10 μl 10x PCR-Puffer10 μl of 10x PCR buffer
2 μl dNTP (je 1OmM im Gemisch)2 μl of dNTP (per 10 mM in the mixture)
5 μl Primer 1 (25pmol)5 μl primer 1 (25 pmol)
5 μl primer 2 (25pmol)) 1 ,5 μl Taq-Polymerase5 μl primer 2 (25 pmol)) 1.5 μl Taq polymerase
74 μl H2O (bidestilliert, steril) auf 100 μl Gesamtvolumen
PCR-Protokoll: 4 min für 94°C. Dann 35 Zyklen mit 1 min für 94°C, 2 min für 55°C und 3 min für 72°C. Abschließend 8 min für 72°C, dann 4°C bis zur Weiterverwendung.74 μl H2O (bidistilled, sterile) to 100 μl total volume PCR protocol: 4 min for 94 ° C. Then 35 cycles of 1 min for 94 ° C, 2 min for 55 ° C and 3 min for 72 ° C. Finally 8 min for 72 ° C, then 4 ° C until further use.
Das PCR-Produkt wird per Gelelektrophorese kontrolliert, aufreinigt und anschließend als PCR-Produkt sequenziert.The PCR product is monitored by gel electrophoresis, purified and then sequenced as a PCR product.
Beispiel 5: Quantifizierung der PromotoraktivitätExample 5: Quantification of promoter activity
Für die quantitative Aktivitätsbestimmung wird als Substrat der ß-Glucuronidase MUG (Methylumbelliferylglucuronid) verwendet, das in MU (Methylumbelliferon) und Glucu- ronsäure gespalten wird. Unter alkalischen Bedingungen kann diese Spaltung quantitativ fluorometrisch verfolgt werden (Anregung bei 365 nm, Messung der Emission bei 455 nm; SpectroFluorimeter Thermo Life Sciences Fluoroscan) wie beschrieben (Bu- stos MM et al. (1989) Plant Cell 1 :839-853).For the quantitative determination of activity, the substrate used is the β-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). ,
Zur Messung der GUS Enzymaktivität wurden 25 mg Pflanzengewebe gemörsert und mit Extraktionspuffer (5OmM Na Phosphat, pH 7; 1OmM Mecaptoethanol; 1OmM EDTA; 0,1% Triton) gemischt. Das unlösliche Pflanzenmaterial wurde durch Zentrifugation (10000g; 10min) sedimentiert. Je 10μl des Überstandes wurden in Multititerplatten zur Messung der GUS-Enzymaktivität vorgelegt. Nach der Zugabe von 90μl Reaktionspuffer (Extraktionspuffer + 2mM Methylumbelliferyl-ß-D-glucuronid) wurde die Entstehung von Methylumbelliferon (MU) pro Minute fluorimetrisch (Anregungswellenlänge: 320nm; Emissionswellenlänge: 405nm) im Bezug zu einer Eichreihe von 10 bis 5000 pmol MU bestimmt. Die Werte wurden auf die nach Bradford bestimmte Proteinmenge bezogen.
To measure 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). 10μl each of the supernatant were presented in multi-well plates for the measurement of GUS enzyme activity. After addition of 90 μl of reaction buffer (extraction buffer + 2 mM methylumbelliferyl-β-D-glucuronide), the formation of methylumbelliferone (MU) per minute was determined fluorimetrically (excitation wavelength: 320 nm, emission wavelength: 405 nm) in relation to a calibration series from 10 to 5000 pmol MU , The values were based on the amount of protein determined according to Bradford.
Claims
1. Verfahren zur gezielten, transgenen Expression von Nukleinsäuresequenzen in1. A method for the targeted, transgenic expression of nucleic acid sequences in
Blütengeweben von Pflanzen, wobei nachfolgende Schritte umfasst sindFlower tissues of plants, wherein subsequent steps are included
I. Einbringen einer transgenen Expressionskassette in pflanzliche Zellen, wobei die transgene Expressionskassette mindestens nachfolgende Elemente enthältI. introducing a transgenic expression cassette into plant cells, wherein the transgenic expression cassette contains at least the following elements
a) mindestens eine Promotorsequenz ausgewählt aus der Gruppe vona) at least one promoter sequence selected from the group of
Sequenzen bestehend ausConsisting of sequences
i) den Promotorsequenzen gemäß SEQ ID NO: 1 , SEQ ID NO: 4,i) the promoter sequences according to SEQ ID NO: 1, SEQ ID NO: 4,
SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undSEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
ii) funktionellen Äquivalenten der Promotorsequenzen gemäß SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 und SEQ ID NO: 9 mit im wesentlichen der gleichen Pro- motoraktivität wie ein Promotor gemäß SEQ ID NO: 1 , SEQ IDii) functional equivalents of the promoter sequences according to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9 with essentially the same promoter activity as a promoter according to SEQ ID NO: 1, SEQ ID
NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 undNO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
iii) funktionell äquivalenten Fragmenten der Sequenzen unter i) oder ii) mit im wesentlichen der gleichen Promotoraktivität wie eineiii) functionally equivalent fragments of the sequences under i) or ii) having substantially the same promoter activity as one
Promotorsequenz gemäß i) oder ii)Promoter sequence according to i) or ii)
undand
b) mindestens eine weitere Nukleinsäuresequenz, undb) at least one further nucleic acid sequence, and
c) gegebenenfalls weitere genetische Kontrollelemente,c) where appropriate, other genetic control elements,
wobei mindestens eine Promotorsequenz und eine weitere Nukleinsäurese- quenz funktionell miteinander verknüpft sind und die weitere Nukleinsäuresequenz in Bezug auf die Promotorsequenz heterolog ist, undwherein at least one promoter sequence and one further nucleic acid sequence are functionally linked to one another and the further nucleic acid sequence is heterologous with respect to the promoter sequence, and
II. Auswahl von transgenen Zellen, die besagte Expressionskassette stabil in das Genom integriert enthalten, undII. Selection of transgenic cells containing said expression cassette stably integrated into the genome, and
4 Fig./Seq IM. Regeneration von vollständigen Pflanzen aus besagten transgenen Zellen, wobei mindestens eine der weiteren Nukleinsäuresequenz in im wesentlichen allen Blütengeweben exprimiert wird.4 Fig./Seq 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.
2. Verfahren zur Identifikation und/oder Isolation von Promotoren von Genen, die für einen Promotor mit Spezifität für Blütengewebe kodieren, wobei bei der Identifikation und/oder Isolation mindestens eine Nukleinsäuresequenz oder ein Teil derselben zum Einsatz kommt, wobei besagte Nukleinsäuresequenz für eine Aminosäuresequenz kodiert, die mindestens einen Teil der Sequenzen gemäß Acc. No. NP 198322, NP 568418, NP 173985, NP 195236, NP 187079, NP 568655 oder eine Variation dieser Sequenzen umfasst.2. A method for the identification and / or isolation of promoters of genes coding for a promoter with specificity for flower tissue, wherein in the identification and / or isolation at least one nucleic acid sequence or a part thereof is used, said nucleic acid sequence encoding an amino acid sequence containing at least part of the sequences according to Acc. No. NP 198322, NP 568418, NP 173985, NP 195236, NP 187079, NP 568655 or a variation of these sequences.
3. Verfahren nach Anspruch 2, wobei das Verfahren unter Einsatz der Polymerase- kettenreaktion durchgeführt wird und die besagte Nukleinsäuresequenz oder ein Teil derselben als Primer eingesetzt wird.3. The method of claim 2, wherein the method is carried out using the polymerase chain reaction and said nucleic acid sequence or a part thereof is used as a primer.
4. Verfahren zur Herstellung einer transgenen Expressionskassette mit Spezifität für Blütengewebe, umfassend nachfolgende Schritte:4. A method for producing a transgenic expression cassette with specificity for flower tissue, comprising the following steps:
I. Isolation eines Promotors mit Spezifität für Blütengewebe, wobei bei der Isolation mindestens eine Nukleinsäuresequenz oder ein Teil derselben zum Einsatz kommt, wobei besagte Nukleinsäuresequenz für eine Aminosäuresequenzen kodiert, die mindestens eine Sequenz gemäß Acc. No. NP 198322, NP 568418, NP 173985, NP 195236, NP 187079, NP 568655 oder eine Variation dieser Sequenzen umfasst;I. 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 sequences containing at least one sequence according to Acc. No. NP 198322, NP 568418, NP 173985, NP 195236, NP 187079, NP 568655, or a variation of these sequences;
II. funktionelle Verknüpfung besagten Promotors mit einer weiteren Nukleinsäuresequenz, wobei besagte Nukleinsäuresequenz in Bezug auf den Promotor heterolog ist.II. Functional linkage of said promoter with another nucleic acid sequence, said nucleic acid sequence being heterologous with respect to the promoter.
5. Verfahren nach Anspruch 4, wobei das Verfahren unter Einsatz der Polymerase- kettenreaktion durchgeführt wird und die besagte Nukleinsäuresequenz oder ein Teil derselben als Primer eingesetzt wird.5. The method of claim 4, wherein the method is carried out using the polymerase chain reaction and said nucleic acid sequence or a part thereof is used as a primer.
6. Transgene Expressionskassette zur gezielten, transgenen Expression von Nu- kleinsäuresequenzen in Blütengeweben von Pflanzen, umfassend6. Transgenic expression cassette for the targeted, transgenic expression of nucleic acid sequences in flower tissues of plants, comprising
a) mindestens eine Promotorsequenz ausgewählt aus der Gruppe von Sequenzen bestehend ausa) at least one promoter sequence selected from the group of sequences consisting of
i) den Promotorsequenzen gemäß SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 und ii) funktionellen Äquivalenten der Promotorsequenzen gemäß SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 und SEQ ID NO: 9 mit im wesentlichen der gleichen Promotoraktivität wie ein Promotor gemäß SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:i) the promoter sequences according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and ii) functional equivalents of the promoter sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9 having substantially the same promoter activity as one Promoter according to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:
7, SEQ ID NO: 10, SEQ ID NO: 11 und SEQ ID NO: 12 und7, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and
iii) funktionell äquivalenten Fragmenten der Sequenzen unter i) oder ii) mit im wesentlichen der gleichen Promotoraktivität wie eine Promotor- sequenz gemäß i) oder ii)iii) functionally equivalent fragments of the sequences under i) or ii) with essentially the same promoter activity as a promoter sequence according to i) or ii)
undand
b) mindestens eine weitere Nukleinsäuresequenz, undb) at least one further nucleic acid sequence, and
c) gegebenenfalls weitere genetische Kontrollelemente,c) where appropriate, other genetic control elements,
wobei mindestens eine Promotorsequenz und eine weitere Nukleinsäuresequenz funktionell miteinander verknüpft sind und die weitere Nukleinsäuresequenz in Bezug auf die Promotorsequenz heterolog ist.wherein at least one promoter sequence and another nucleic acid sequence are functionally linked together and the further nucleic acid sequence is heterologous with respect to the promoter sequence.
7. Transgene Expressionskassette nach Anspruch 6, wobei7. Transgenic expression cassette according to claim 6, wherein
a) die zu exprimierende Nukleinsäuresequenz mit weiteren genetischen Kon- trollsequenzen funktionell verknüpft ist, odera) the nucleic acid sequence to be expressed is functionally linked to further genetic control sequences, or
b) die Expressionskassette zusätzliche Funktionselemente enthält, oderb) the expression cassette contains additional functional elements, or
c) a) und b) gegeben sind.c) a) and b) are given.
8. Transgene Expressionskassette nach Anspruch 6 oder 7, wobei die transgen zu exprimierende Nukleinsäuresequenz8. Transgenic expression cassette according to claim 6 or 7, wherein the transgene to be expressed nucleic acid sequence
a) die Expression eines von besagter Nukleinsäuresequenz kodierten Prote- ins, odera) the expression of a protein encoded by said nucleic acid sequence, or
b) die Expression eines von besagter Nukleinsäuresequenz kodierter sense- RNA, anti-sense-RNA oder doppelsträngigen RNA ermöglicht.b) allows the expression of a sense RNA, antisense RNA or double-stranded RNA coded by said nucleic acid sequence.
9. Transgene Expressionskassette nach einem der Ansprüche 14 bis 17, wobei die transgen zu exprimierende Nukleinsäuresequenz ausgewählt ist aus der Gruppe von Nukleinsäuresequenzen kodierend für Chalconsynthasen, Phenylalaninam- moniumlyasen, Photolyasen, Deoxyxylulose-5-phosphatsynthasen, Phytoen- synthasen, Phytoendesaturasen, Lycopincyclasen, Hydroxylasen, "antifreeze"- Polypeptide, CBF1 -Transkriptionsaktivatoren, Glutamatdehydrogenasen, Calci- um-abhängigen Proteinkinasen, Calcineurin, Famesyltransferasen, Ferritin, Oxa- latoxidasen, DREBIA-Faktor, Trehalosephosphatphosphatasen, Chitinasen, GIu- canasen, Ribosom-inaktivierende Protein, Lysozyme, Bacillus thuringiensis Endo- toxine, Amylaseinhibitoren, Proteaseinhibitoren, Lectinen, RNAsen, Ribozymen, Endochitinase, Cytochrom P-450, Acetyl-CoA Carboxylasen, Aminosäure- Transporter, Monosaccharid-Transporter, Lycopincyklasen, Carotinketolasen, En- doxyloglucantransferasen, Δ6-Acyllipiddesaturasen, Δ6-Desaturasen, Δ5-9. Transgenic expression cassette according to one of claims 14 to 17, wherein the nucleic acid sequence to be expressed transgenically is selected from the group of nucleic acid sequences coding for chalcone synthases, phenylalanine amino monolylases, photolyases, deoxyxylulose-5-phosphate synthases, phytoene synthases, phytoene desaturases, lycopene cyclases, hydroxylases, antifreeze polypeptides, CBF1 transcriptional activators, glutamate dehydrogenases, calcium-dependent protein kinases, calcineurin, famesyltransferases, ferritin, oxalate oxidases, DREBIA Factor, Trehalose Phosphate Phosphatases, Chitinases, Gulanases, Ribosome Inactivating Protein, Lysozymes, Bacillus thuringiensis Endotoxins, Amylase Inhibitors, Protease Inhibitors, Lectins, RNAses, Ribozymes, Endochitinase, Cytochrome P-450, Acetyl-CoA Carboxylases, Amino Acid Transporters, Monosaccharide transporters, lycopene cyclases, carotene tolases, endo-glucan transferases, Δ6-acyl lipid desaturases, Δ6-desaturases, Δ5-
Fettsäuredesaturase, Δ6-Elongasen und IPP-lsomerasen.Fatty acid desaturase, Δ6 elongases and IPP isomerases.
10. Transgene Expressionskassette nach einem der Ansprüche 6 bis 9, wobei die transgen zu exprimierende Nukleinsäuresequenz ausgewählt ist der Gruppe von Nukleinsäuresequenzen beschrieben durch GenBank Acc.-No.: M20308,10. A transgenic expression cassette according to any one of claims 6 to 9, wherein the nucleic acid sequence to be expressed transgenically is selected from the group of nucleic acid sequences described by GenBank Acc. No .: M20308,
BAB00748, U62549, U77378, S78423, U32624, L25042, X92657, AJ002399, D45881 , AF163819, AB044391 , AJ222980 und AF078796.BAB00748, U62549, U77378, S78423, U32624, L25042, X92657, AJ002399, D45881, AF163819, AB044391, AJ222980 and AF078796.
11. Transgener Expressionsvektor enthaltend eine Expressionskassette gemäss ei- nem der Ansprüche 6 bis 10.11. Transgenic expression vector containing an expression cassette according to any one of claims 6 to 10.
12. Transgener Organismus transformiert mit einer Expressionskassette gemäß den Ansprüchen 6 bis 10 oder einem Expressionsvektor gemäß Anspruch 11.12. Transgenic organism transformed with an expression cassette according to claims 6 to 10 or an expression vector according to claim 11.
13. Transgener Organismus nach Anspruch 12 ausgewählt aus der Gruppe bestehend aus Bakterien, Hefen, Pilzen, nicht-menschlichen tierischen und pflanzlichen Organismen oder von diesen abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut.13. A transgenic organism according to claim 12 selected from the group consisting of bacteria, yeasts, fungi, non-human animal and vegetable organisms or derived from these cells, cell cultures, parts, tissues, organs or reproductive material.
14. Transgener Organismus nach Anspruch 12 oder 13 ausgewählt aus der Gruppe der landwirtschaftlichen Nutzpflanzen.14. Transgenic organism according to claim 12 or 13 selected from the group of agricultural crops.
15. Verwendung eines transgenen Organismus nach einem der Ansprüche 12 bis 14 oder von diesem abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut zur Herstellung von Nahrungs-, Futtermitteln, Saatgut, Pharma- zeutika oder Feinchemikalien.15. Use of a transgenic organism according to any one of claims 12 to 14 or derived therefrom cells, cell cultures, parts, tissues, organs or propagation material for the production of food, feed, seeds, pharmaceuticals or fine chemicals.
16. Verfahren zur Herstellung von Pharmazeutika oder Feinchemikalien in transgenen Organismen nach einem der Ansprüche 12 bis 14 oder von diesem abgeleitete abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut, wobei der transgene Organismus oder von diesem abgeleitete abgeleitete Zellen, Zellkulturen, Teile, Gewebe, Organe oder Vermehrungsgut gezüchtet oder kulti- viert werden und das gewünschte Pharmazeutikon oder die gewünschte Fein- chemikalie isoliert werden. 16. A process for the preparation of pharmaceuticals or fine chemicals in transgenic organisms according to any one of claims 12 to 14 or derived derived cells, cell cultures, parts, tissues, organs or propagation material, wherein the transgenic organism or derived therefrom derived cells, cell cultures, parts , Tissues, organs or reproductive material or cultivated and the desired pharmaceutical or the desired fine chemical are isolated.
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PCT/EP2006/062012 WO2006117381A2 (en) | 2005-05-04 | 2006-05-03 | Transgenic expression cartridges for expressing nucleic acids in the flower tissue of plants |
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CN116042694A (en) * | 2022-11-24 | 2023-05-02 | 中国科学院南京土壤研究所 | Non-tissue culture genetic transformation method for pennisetum plants of Gramineae |
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EP0913469B1 (en) * | 1996-12-27 | 2008-07-02 | Japan Tobacco Inc. | Floral organ-specific promoter sequences |
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 |
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CA2496300A1 (en) * | 2002-08-20 | 2004-04-01 | Sungene Gmbh & Co. Kgaa | Transgenic expression cassettes for the expression of nucleic acids in plant blooms |
EP1606304A4 (en) * | 2003-03-12 | 2006-09-27 | Evogene Ltd | Nucleotide sequences regulating gene expression and constructs and methods utilizing same |
DE102004007623A1 (en) * | 2004-02-17 | 2005-08-25 | Sungene Gmbh & Co. Kgaa | Use of specific promoters for expressing genes in Tagetes, useful for preparing biosynthetic products, specifically carotenoids, for use as e.g. pharmaceuticals, also the genetically modified plants |
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