DE10053519A1 - Expression cassette containing plant myb11 promoter, useful for preparing transgenic plants, provides specific expression in flowers and embryonal epidermis - Google Patents

Abstract

Expression cassette (EC) for transgenic expression of nucleic acid (NA) comprises the promoter of the myb11 gene from Arabidopsis thaliana (2964 bp sequence (1), reproduced), its functional equivalent or fragments with essentially the same promoter activity. Independent claims are also included for the following: (1) vector containing EC; (2) transgenic organism transformed with EC or the vector of (1); and (3) cell cultures, parts and transgenic replicative material from the organisms of (2).

Description

The invention relates to expression cassettes and vectors that plant promoters with an expression specificity for the embryonic epidermis and / or the flower or functionally equiva lent parts of the same, as well as the use of these Expression cassettes or vectors for the transgenic expression of Nucleic acid sequences in transgenic organisms, preferably in Plants. The invention further relates to these expressions cassettes or vectors transformed transgenic plants thereof derived cultures, parts or transgenic reproductive material, as well as the use of the same for the production of food, feed agents, seeds, pharmaceuticals or fine chemicals.

Different methods of introducing genes into the genome of Plants are known (Halford NG, Shewry PR, Br Med Bull 2000; 56 (1): 62-73). The aim is to produce plants with beneficial adhere to new properties for example to increase land economic productivity, to improve quality at Nah foodstuffs or for the production of certain chemicals or Pharmaceuticals (Dunwell JM, J Exp Bot. 2000; 51 Spec No: 487-96). Often the natural defense mechanisms of the plant are part of it inadequate play against pathogens. The introduction of foreign genes The defense can come from plants, animals or microbial sources strengthen. Examples are the protection against insect caused damage in Ta bak by expressing the Bacillus thuringiensis endotoxin Control of the 35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37) or the protection of tobacco against fungal attack by Ex pression of a chitinase from the bean under the control of the CaMV Promoters (Broglie et al., Science 1991, 254, 1194-1197). Further can become herbicide resistance through the introduction of foreign genes aims to optimize the growing conditions and reduce the harvest ste decreased (Ott KH et al., J Mol Biol. 1996; 263 (2): 359-368). The quality of the products can also be improved. So can the shelf life and storability of harvested products game can be increased by inactivating certain maturation genes. This was shown, for example, on the tomato through inactivation of polygalacturonase (Hamilton AJ et al., Curr Top Microbiol Im munol 1995; 197: 77-89).

A basic requirement for the transgenic expression of certain Genes is the provision of plant-specific promoters. Pro motors are important tools in plant biotechnology the expression of a particular gene in a transgenic plant to control and thus to educate certain essential characteristics of the plant len. Various plant promoters are known.

For example, constitutive promoters such as the Promo are known tor of nopaline synthase from Agrobacterium, the TR double promoter or the promoter of the 35S transcript of the cauliflower mosaic virus (CaMV) (Odell et al., Nature 313: 810-812 (1985)), the OCS (Octo pin synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf 5 et al., Plant Mol Biol 1995, 29: 637-646), the Promoto ren of the vacuolar ATPase subunits or the promoter of a wheat proline-rich protein (WO 91/13991). Disadvantageous with these promoters is that they can be found in almost all tissues of the plant are constitutively active. A targeted expression of genes in be correct parts of the plant or at a certain point in time of development ten is not possible with these promoters.

Promoters with specificities for the anthers, ovaries, flowers, Leaves, stems, roots and seeds are described. The Strin the specificity as well as its expression activity Promoters is very different. Mention should be made of promoters, which ensure leaf-specific expression, like the Pro motor of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of Rubisco (Ribulose-1,5-bisphosp hatcarboxylase) or the ST-LSI promoter from potatoes (Stockhaus et al., 1989, EMBO J. 8: 2445-245).

Further promoters are, for example, promoters with specificity for tubers, storage roots or roots such as the Patatin promoter class I (B33), the promoter of cathepsin D In potato hibitors, the promoter of starch synthase (GBSS1) or the sporamine promoter, fruit-specific promoters such as for example the fruit-specific promoter from tomatoes (EP-A 409625), fruit ripening-specific promoters, as in for example the fruit ripening-specific promoter from tomatoes (WO 94/21794), flower-specific promoters such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).

A variation in the activity of a promoter depending on the Ent development stage of the plant is among others in Baerson et al. (Baerson SR, Lamppa GK. Plant Mol Biol. 1993; 22 (2): 255-67).

Also known are promoters that allow expression in seeds and control plant embryos. For example, the Pro identified motors of genes responsible for storage proteins ver of different dicotyledons. Seed Specific Promoters are for example the promoter of phaseolin (US 5,504,200, Bustos mm et al., Plant Cell. 1989; 1 (9): 839-53), des 25 Albumin gens (Joseffson LG et al., J Biol Chem 1987, 262: 12196-12201), of legumin (Shirsat A et al., Mol Gen Genet. 1989; 215 (2): 326-331), the USP (unknown seed protein; Bäumlein H. et al., Molecular & General Genetics 1991, 225 (3): 459-67) des Napin Gens (Stalberg K, et al., L. Planta 1996, 199: 515-519), des Sucrose binding protein (WO 00/26388) or the LeB4 promoter (Baumlein H et al., Mol Gen Genet 1991, 225: 121-128). These control a seed-specific expression of storage proteins.

The level of transgenic expression of heterologous genes under However, control of these promoters is often strong in nature Host plant dependent. It was also found that the Expression is rarely cell type specific. Differences in the Expres sion pattern and the level of expression of a particular promoter can by different host plants or by under different insertion sites in the genome of the host plant (Goossens A et al., Plant Phys 1999, 120: 1095-1104).

A promoter activity with a specificity for certain cells inside the seed is only described for the seed coat been: A cryptic promoter with specificity for the seeds shell was identified by "T-DNA tagging" in tobacco (Fobert PR et al., Plant Journal 19946 (4): 567-77; U.S. 5,824,863; WO 99/53067). Cryptic promoters or pseudo promoters are on are inactive sequences within the genome, but which are a Get expression-regulating functionality if it is in the Be positioned close to genes.

The plant epidermis provides both the primary barrier as well also the essential contact surface of the plant organism its environment. It plays an important role in protecting it of the plant against biotic and ablotic stressors, however also in the absorption of nutrients.

The epidermis-specific expression of certain genes is known. So is a lectin-like protein that is found in the sprout spit zenepidermis of the garden pea (Pisum sativum) enriches, be (Maiti et al., Planta 1993, 190, 241-246). Sterk et al. describe a lipid transfer protein that is potent in the epiderma len cells are expressed by the tips of the carrot shoots (Sterk et al., Plant Cell 1991, 3, 907-921). Clark et al. (Clark et al., Plant Cell 1992, 4, 1189-1198) describe a lipid transfer pro tein that is expressed in the epidermis of Pachyphytum. White Further examples of epidermis-specifically expressed genes, such as Chalcone synthase and phenylalanine ammonium yase gene families reported (Schmelzer et al., Proc. Natl. Acad. Sci. USA 1988, 85, 2989-2993; Schmelzer et al., Plant Cell 1989, 1, 993-1001). Goo drich et al. describes enzymes involved in anthocyanin biosynthesis in the Snapdragon flower residing in specialized, epidermal cells for a limited period of time during the development of the flowers nospe (Goodrich et al., Cell 1992, 68, 955-964). Watt et al. describes the proline-rich cell wall pro teine SbPRP1, SbPRP2, and SbPRP2 from soy, which lead to certain Ent stages of development in the epidermis, but also in the Vascular tissue are expressed (Wyatt et al., Plant Cell 1992, 4, 99-110).

Promoters are described with tissue specificity for the meso phyll and the pallisade cells in leaves (Broglie et al., Science 1984, 234, 838-845), the dividing scion and the Root meristem (Atanassova et al., Plant J. 1992, 2, 291-300), Pollen (Guerrero et al., Mol. Gen. Genet. 1990, 224, 161-168), Sa menendosperm (Stalberg et al., Plant Mol. Biol. 1993, 23, 671-683), root epidermis (Suzuki et al., Plant Mol. Biol 1993, 21, 109-229), as well as for the root meristem, root vascular tissue and root nodules (Bogusz et al., Plant Cell 1990, 2, 633-641).

There are also known epidermis-specific promoters with Akti vity in flowers (Koes et al., Plant Cell 1990, 2, 379-392) or after wound injuries (Liang et al., Proc. Natl. Acad. Sci. USA 1989, 86, 9284-9288). US 5,744,334 describes a vegetable Promoter (Blec promoter) with specificity for the epidermis of the Sprout tip. In contrast, no promoter has yet been described that specifically the expression in the plant, embryonic epi dermis guaranteed.

The expression of genes is in all organisms via transcript regulating factors. These also show developmental and location-dependent expression patterns. Of particular interest is the Family of R2R3-MYB transcription factors that are conserved DNA binding domains comparable to that of the transcription factor c-myb and have been identified in almost all eukaryotes the. It is believed that Arabidopsis thaliana has more than 90 R2R3-MYB has genes (Meissner RC et al., Plant Cell 1999; 11 (10): 1827-1840).

Despite the large number of known plant promoters, up long no promoter with a specificity for the epidermis of the plant embryos identified. Also there are no promoters known to have an expression in the flower and in the epidermis of the plant embryo.

The task was therefore to identify appropriate promoters. This object was achieved by providing the promoter for the myb11 gene from Arabidopsis thaliana. The promoter of the myb11 gene from Arabidopsis thaliana has an expression-regulating specificity that shows expression in the flower bud, the flower, the wilting flower, the green seed pods and in the embryonic epidermis on day 4 of germination (compare FIGS. 1 to 6 ).

The myb11 gene belongs to the R2R3-MYB family of Transkriptionfak fools. The MYB11 gene is known (Genbank Acc.-No: AL162651). However, neither its function nor its expression pattern are be wrote. So far, no MYB transcription factor has been described or has been identified that both in flowers and in the embryonic epidermis or alone in the embryonic epidermis is expressed.

The expression specificity of expression cassettes derived from this promoter in the flower and the embryonic epidermis is particularly advantageous because

• a) the flower bud and the flower of the plant a delicate one Organ, especially against stress factors such as cold, is and
• b) the plant embryo especially in the first days of Em bryogenesis particularly sensitive and susceptible to biotic and is ablotic stressors.

A particularly funk occurs to the epidermis of the plant embryo tion, as it is the immediate barrier and point of contact to Represents the environment.

A first object of the invention therefore relates to an expression cassette for the transgenic expression of nucleic acids in the embryonic epidermis and / or the flower

• a) the promoter of the mybll gene from Arabidopsis thaliana according to SEQ ID NO: 1, or
• b) a functional equivalent or equivalent fragment of a) that have essentially the same promoter activities as a) own,

where a) or b) functionally with a transgene to be expressed Nucleic acid sequence are linked.

Expression cassette for the transgenic expression of nucleic acids means all such constructions that have come about by genetic engineering methods in which either

• a) the promoter with specificity for the embryonic epidermis and / or the flower or a functional one derived from it Equivalent or equivalent fragment thereof, or
• b) the nucleic acid sequence to be expressed, or
• c) (a) and (b)

not in their natural, genetic environment (i.e. their natural chromosomal locus) or by gen technical methods were modified, the modification exemplary a substitution, additions, deletions, inversions sion or insertions of one or more nucleotide residues can.

In the context of the expression cassette according to the invention, the Ex pression of a particular nucleic acid by a promoter Specificity for the embryonic epidermis and / or the flower too Formation of sense RNA or anti-sense RNA lead. The sense RNA can consequently be translated into certain polypeptides. With The anti-sense RNA can result in the expression of certain genes be regulated.

A functional link is understood to mean, for example the sequential arrangement of the promoter with specificity for the embryonic epidermis and / or the flower transgenic to ex priming nucleic acid sequence and possibly other regulatory ones Elements such as a terminator such that each of the regulatory elements its role in transgenic expression the nucleic acid sequence, depending on the arrangement of the nucleic acid sequence sequences to sense or anti-sense RNA. Is to not necessarily a direct link in the chemical sense conducive. Genetic control sequences such as Enhan cer sequences can function from further away Positions or even from other DNA molecules on the target exercise sequence. Arrangements are preferred in which the transgenic nucleic acid sequence to be expressed behind the fun yawing sequence is positioned so that both sequences are covalently linked. The Ab is preferred stood between the promoter sequence and the transgenic expression generating nucleic acid sequence less than 200 base pairs, especially preferably less than 100 base pairs, very particularly preferred less than 50 base pairs.

The creation of a functional link can be done by means of gän giger recombination and cloning techniques can be implemented, as described, for example, in T. Maniatis, E. F. Fritsch and J. Sam brook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987) ben are. However, further Se can also be used between the two sequences sequences that, for example, have the function of a Linkers with certain restriction enzyme sites or one Have signal peptides. The insertion of sequences for Lead expression of fusion proteins.

Various ways are known to those skilled in the art to obtain an invented to arrive according to the expression cassette. The production an expression cassette according to the invention takes place, for example wise preferably by direct fusion of a promoter with Spe specificity for the embryonic epidermis and / or the flowering fungia generating nucleic acid sequence with a nucleotide to be expressed sequence. Common recombination and cloning are used for this purpose tion techniques, such as those described in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) supra as in Silhavy, T.J., Berman, M.L., and Enquist, L.W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Inter science (1987). Preferably the expression cassette, consisting of a link between promoter and ex priming nucleic acid sequence integrated in a fiction according to the vector and by, for example, transformation inserted into a plant genome.

However, such constructs are also included under an expression cassette tions to understand where the promoter without him beforehand with a nucleic acid sequence to be expressed functionally ver was linked, for example via a targeted homologous recombina tion or a random insertion into a host genome then there will be regulatory control over with it functional linked nucleic acid sequences takes over and the transgenic Ex pression of the same controls. By inserting the promoter - to Example through a homologous recombination - in front of a for a be A nucleic acid encoding the correct polypeptide is obtained Expression cassette according to the invention, which the expression of the be agreed polypeptides selectively in the embryonic epidermis and / or the bloom controls. Furthermore, the insertion of the promoter also take place in such a way that antisense RNA to the for a certain tes polypeptide-encoding nucleic acid is expressed. In order to will selectively limit the expression of the particular polypeptide in the embryonic epidermis and / or the flower downregulated or switched off.

A nucleic acidesis to be expressed transgenically can also be analogous sequence, for example, by homologous recombination behind the endogenous, natural myb11 promoter are placed, whereby an expression cassette according to the invention is obtained which contains the Expression of the nucleic acid sequence to be expressed transgenically in controls the embryonic epidermis and / or the flower.

A promoter activity is essentially referred to as the same net if the transcription of any to be expressed Gene under the control of a particular one derived from SEQ ID NO: 1 th promoter in at least one of the two target compartments expression (flower or embryonic epiderm) takes place. Included the level of expression can be downwards as well as upwards in the Compared to a comparative value differ. Are preferred those sequences whose level of expression, measured using the transcribed mRNA or the protein translated as a result, un the otherwise unchanged conditions quantitatively by no more than 50%, preferably 25%, particularly preferably 10% of a ver obtained equivalent to the Pro described by SEQ ID NO: 1 engine differs. Particularly preferred are those sequences their level of expression, measured on the basis of the transcribed mRNA or the protein translated as a result, under otherwise unchanged changed conditions quantitatively by more than 50%, preferably 100%, particularly preferably 500%, very particularly preferably 1000% Comparative value obtained with that described by SEQ ID NO: 1 Promoter exceeds. The expression is preferred as a comparison value sion level of the natural myb11 mRNA or the natural MYB11 Arabidopsis thaliana protein. Is also preferred as Ver equivalently the level of expression obtained with any, but certain nucleic acid sequence, preferably such nucleus acid sequences that code for easily quantifiable proteins. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) like that "green fluorescence protein" (GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23 (5): 912-8, 1997), the chloramphenicol transferase, a luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414) or the β-galactosidase, β-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907).

Otherwise unchanged conditions means that the expression, by one of the expression cassettes to be compared in is itiated, not by combining it with additional geneti control sequences, for example enhancer sequences, modified is adorned.

According to the invention are also vectors which are those described above Expression cassettes included.

Those in the expression cassettes or vectors according to the invention contained nucleic acid sequences can with further genetic Control sequences in addition to the promoter with specificity for the em bryonic epidermis and / or the flower be functionally linked. The term genetic control sequences can be used broadly and means all those sequences that have an influence on the Origin or function of the expressions according to the invention have sion cassette. Modify genetic control sequences for example transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the inven according to the expression cassettes 5 'upstream of the ever particular nucleic acid sequence to be expressed transgenically the promo tor with specificity for the embryonic epidermis and / or the Flower and 3'-downstream a terminator sequence as an additional genetic control sequence and, if applicable, other customary ones regulative elements, each functionally linked to the nucleic acid sequence to be expressed transgenically.

Genetic control sequences also include other promoters, Promoter elements or minimal promoters that control the expression can modify the properties. So through geneti cal control sequences, for example the tissue-specific express sion also take place depending on certain stress factors. Corresponding elements are for example for water stress, absci sic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131-17135) and heat stress (Schöffl F et al., Molecular & General Genetics 217 (2-3): 246-53, 1989).

Further promoters can also be functional with the to be expri mating nucleic acid sequence linked to an expression in other plant tissues or in other organisms, such as Example E. coli bacteria enable. As plant promoters In principle, all of the promoters described above are suitable. It is conceivable, for example, that a certain nucleic acid synthesis sequence by a promoter (for example the promoter with speci for the embryonic epidermis and / or the flower) in one Transcribed plant tissue as sense RNA and into the corresponding The corresponding protein is translated while the same nucleic acid sequence by a different promoter with a different specifi ity is transcribed to anti-sense RNA in another tissue and the corresponding protein is downregulated. This can realized by an expression cassette according to the invention who by placing the one promoter in front of the transgene to be expressed Nucleic acid sequence is positioned and the other promoter there Behind.

Genetic control sequences also include the 5'-untrans latent region, introns, or the 3 'non-coding region of Genes, preferably the mybll gene from Arabidopsis thaliana. It is this has been shown to play a significant role in the Regulation of gene expression can play. So it was shown that 5 'untranslated sequences hete transient expression rologer genes can amplify. You can also use the tissue sp promote ciity (Rouster J et al., Plant J. 1998, 15: 435-440.). Conversely, the 5 'untranslated region of the opaque-2 suppresses Gene expression. A deletion of the corresponding region leads to an increase in gene activity (Lohmer S et al., Plant Cell 1993, 5: 65-73). The nucleic acid given under SEQ ID NO: 1 The sequence contains the section of the mybll gene that contains the promoter and the 5 'untranslated region before the ATG start codon des MYB11 transcription factor.

The expression cassette can advantageously one or more so-called "enhancer sequences" functionally linked to the Contain promoter that increases transgenic expression of the Nu allow small acid sequence. Also at the 3 'end of the transgenic too expressing nucleic acid sequences can provide additional benefit Adherent sequences are inserted, such as further regulatory ele ments or terminators. The nucleus to be expressed transgenically acid sequences can be in one or more copies in the gene con be included.

Additional to the functional link preferred but not Sequences restricted to this are further ones from the transit peptide coding sequences different, targeting sequences for Ge ensuring subcellular localization in the apoplast in which Vacuoles, in plastids, in the mitochondrion, in the endoplasmic re tikulum (ER), in the cell nucleus, in oil bodies or other compartments ments; as well as translation enhancers such as the 5 'leader sequence the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711) and the like.

Control sequences are also to be understood as those that a homologous recombination or insertion into the genome of a Host organism enable or remove it from the genome arbor. In homologous recombination, for example, the na natural promoter of a particular gene against a promoter with Specificity for the embryonic epidermis and / or the flower be exchanged. Allow methods like cre / lox technology a tissue-specific, possibly inducible removal the expression cassette from the genome of the host organism (Sauer B. Methods. 1998; 14 (4): 381-92). This is where certain flankies become Ending sequences are added to the target gene (lox sequences), which later allow removal by means of cre recombinase.

The promoter to be introduced can by means of homologous recombination placed in front of the target gene to be expressed transgenically by the promoter is linked to DNA sequences, for example are homologous to endogenous sequences that are in the reading frame of the target gens are upstream. Such sequences are called genetic Understand control sequences. After a cell with the ent speaking DNA construct has been transformed, the two can homologous sequences interact and so the promoter sequence the desired location in front of the target gene so that the Pro motor sequence now functionally linked to the target gene and forms an expression cassette according to the invention. the Selection of the homologous sequences determines the insertion site of the Promoters. The expression cassette can be replaced by homologous Recombination by means of a single or a double reci proken recombination can be generated. With the simply reciprocal Recombination will only use a single recombination sequence and all of the introduced DNA is inserted. In the case of double-reciprocal recombination, the one to be introduced is DNA is flanked by two homologous sequences and the Insertion of the flanked area. The latter method is ge suitable to the natural promoter of a as described above specific gene against the promoter with specificity for the embryo Replace the nal epidermis and / or the flower and thus the ex to modify the place and time of pressure of this gene. the functional linkage is an expression according to the invention sion cassette.

The expression cassettes according to the invention and the vectors derived from them can 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, multiplication or function of the expression cassettes, vectors or transgenic organisms according to the invention. The following are examples, but not restrictive:

• a) Selection markers showing resistance to a metabolis Musin inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), Antibiotics or biocides, preferably herbicides, such as for play Kanamycin, G 418, Bleomycin, Hygromycin, or Phosphi give notricin etc.
• b) Reporter genes that code for easily quantifiable proteins ren and an assessment of their own color or enzyme activity the transformation efficiency or the expression site or - ensure the time. They are especially preferred in reporter proteins (Schenborn E, Groskreutz D. Mol Bio technol. 1999; 13 (1): 29-44) like the "green fluorescence pro tein "(GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Lef fel SM et al., Biotechniques. 23 (5): 912-8, 1997), the Chlo ramphenicoltransferase, a luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414), the β-galactosidase, whole be β-glucuronidase is particularly preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907)
• c) origins of replication, which are an increase in the erfindungsge need expression cassettes or vectors in for example E.coli guarantee. Examples are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sam brook et al .: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) elements necessary for an agrobacterium mediated plants Transformation is required, such as the rights or left border of the T-DNA or the vir region.

For the selection of successfully homologously recombined or trans formed cells it is usually necessary to select a selec In addition to introducing a possible marker, which is successful recombined cells develop resistance to a biocide (for play a herbicide), a metabolism inhibitor like 2-deoxyglu cose-6-phosphate (WO 98/45456) or an antibiotic. Of the Selection marker allows the transformed cells to be selected of untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84).

Homologous recombination is a relatively rare occurrence in high ren eukaryotes, especially in plants. Random integrations in the host genome predominate. One possibility that happens to be inte to remove grated sequences and so cell clones with a cor Enriching direct homologous recombination consists in the Ver use of a sequence-specific recombination system as in US 6,110,736. This consists of three elements: two Pairs of specific recombination sequences and one sequence specific recombinase. This recombinase catalyzes a recombination only between the two pairs of spe specific recombination sequences. The one pair of these specifi different DNA sequences is outside of the DNA-Se to be integrated quenz, d. H. locally outside the two homologous DNA sequences sated. In the case of a correct homologous recombination will be these sequences are not transferred into the genome. In case of a usually insert them together with random integration the rest of the construct. Using a special recombi nose and a construct containing a second pair of spec Fish sequences can produce the randomly inserted sequences cut out or inactivated by inversion while the correctly inserted via homologous recombination in the Genome remain. A variety of sequence-specific recom Bination systems can be used, these are exemplary Cre / lox system of the bacteriophage P1, the FLP / FRT system of the Yeast, the gin recombinase of the Mu phage, the pin recombinase E. coli and the R / RS system of the pSR1 plasmid. Preferred are the bacteriophage P1 Cre / lox and the yeast FLP / FRT system. Here the recombinase (Cre or FLP) interacts specifically with their respective recombination sequences (34bp lox sequence or 47bp FRT sequence) to delete the intermediate sequences ren or invert. The FLP / FRT and cre / lox recombinase stem has already been used in plant systems (Odell et al., Mol. Gen. Genet., 223: 369-378, 1990.)

Suitable control sequences are polyadenylation signals vegetable polyadenylation signals, preferably those im essential T-DNA polyadenylation signals from Agrobacterium tu mefaciens, especially gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents thereof. Examples for particularly suitable terminator sequences, the OCS (Octo pin synthase) terminator and the NOS (nopaline synthase) termina gate.

The expression cassettes according to the invention or removed from them Derived vectors can be functional equivalents to the subordinate SEQ ID NO: 1 described sequence of the promoter of the MYB11 trans of Arabidopsis thaliana. Functional equivalent sequences also include all the sequences identified by the complementary opposite strand defined by SEQ ID NO: 1 Sequence derived and essentially the same promoto have activity.

Functional equivalents initially means DNA sequences that are listed under Standard conditions with that described by SEQ ID NO: 1 Nucleic acid sequence or the nucleic acid complementary to it hybridize sequence and perform essentially the same promo gate activities as described by SEQ ID NO: 1 nucleic acid have sequence. Standard hybridization conditions are broad too understand and mean stringent as well as less stringent hybri pricing conditions. Such hybridization conditions are among others in Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning (A Laboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. described.

For example, the conditions during the washing step can be selected from the range of conditions limited by those with 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) (20X SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature can be increased during the washing step from low stringent 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, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C. Some exemplary conditions for hybridization and washing step are given as a result:

• 1. Hybridization conditions with for example
  • a) 4X SSC at 65 ° C, or
  • b) 6X SSC at 45 ° C, or
  • c) 6X SSC at 68 ° C, 100 µg / ml denatured fish sperm DNA, or
  • d) 6X SSC, 0.5% SDS, 100 µg / ml denatured, fragmented salmon sperm DNA at 68 ° C, or
  • e) 6X SSC, 0.5% SDS, 100 µg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42 ° C.
  • f) 50% formamide, 4X SSC at 42 ° C, or
  • g) 50% (vol / vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42 ° C, or
  • h) 2X or 4X SSC at 50 ° C (weakly stringent condition), or
  • i) 30 to 40% formamide, 2X or 4X SSC at 42 ° C (weakly stringent condition).
• 2. Washing steps with for example
  • a) 0.015 M NaCl / O.0015 M sodium citrate / 0.1% SDS at 50 ° C; or
  • b) 0.1X SSC at 65 ° C, or
  • c) 0.1X SSC, 0.5% SDS at 68 ° C, or
  • d) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C, or
  • e) 0.2X SSC, 0.1% SDS at 42 ° C, or
  • f) 2X SSC at 65 ° C (weakly stringent condition).

The use of such nucleic acids is preferred according to the invention in the expression cassettes or vectors derived from them, with the double-stranded DNA described by SEQ ID NO: 1 Sequence or fragments thereof, such as, for example, those with SEQ ID NO: 2 and 15 to 19 described double-stranded DNA sequences zen, hybridize under the conditions defined above and im have substantially the same promoter activity as.

A functional equivalent is also understood in particular which also include natural or artificial mutations of the under SEQ ID NO: 1 described mybll promoter and its homologues from their plant genera and species, which continue to be an Expres sion in the plant embryonic epidermis and / or flowers can initiate. Mutations include substitutions, additions nes, deletions, inversions or insertions of one or more Nucleotide residues. Thus, for example, such Nucleo tide sequences encompassed by the present invention, which obtained by modifying the mybll promoter nucleotide sequence according to SEG ID NO: 1. The aim of such a modification can e.g. B. the further limitation of the sequence contained therein to be agreed regulatory elements or e.g. B. also the insertion white terer restriction enzyme sites, the removal is unnecessary ger DNA or the addition of further sequences, for example white terer regulatory sequences.

The limitation of the sequence to certain essential regulato Roman regions can also be searched for by using a search routine Promoter elements are made. Often times are in the for the Promoter activity relevant regions specific promoter elements often present. This analysis can, for example, be carried out with Compu ter programs such as the PLACE program ("Plant Cis-acting Regula tory DNA Elements ") (K. Higo et al., (1999) Nu cleic Acids Research 27: 1, 297-300).

Where insertions, deletions or substitutions, such as e.g. B. Trans itions and transversions that come into question can be in themselves known techniques such as in vitro mutagenesis, "primer repair", Re restriction or ligation can be used. Through manipulation, such as B. Restriction, "chewing-back" or filling in overhangs genes for "blunt ends" can have complementary ends of the fragments for the ligation can be provided. To an analogous result One can also sen using the polymerase chain reaction (PCR) using specific oligonucleotide primers.

Homology between two nucleic acids is understood to mean the identity of the nucleic acid sequence over the respective entire sequence length, which is determined by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) under setting fol gender parameter is calculated:

Gap Weight: 12th Length Weight: 4th Average Match: 2.912 Average mismatch: -2.003

By way of example, a sequence which has a homology of at least 20% based on nucleic acid with the sequence SEQ ID NO: 1 has understood a sequence that when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with obi according to the parameter set has a homology of at least 20%.

Functional equivalents derived from one of the in the inven appropriate expression cassettes or vectors are used menden promoter sequences, for example by substitution, insert tion or deletion of nucleotides, have a homology of min at least 20%, preferably 40%, preferably at least 60%, be particularly preferably at least 80%, very particularly preferably min at least 90%, and are characterized by essentially the same che transcriptional activity compared to the myb11 promoter sequence according to SEQ-ID NO: 1.

Further examples of the expressions according to the invention cassettes or expression vectors used promo Gate sequences can, for example, be taken from different org nisms whose genomic sequence is known, such as from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, So lanum tuberosum, Helianthinum, through homology comparisons from Da Find databases easily.

Functional equivalents also include promoter variants their function, compared to the original promoter, weakened or is reinforced.

As a further suitable functionally equivalent expression cassette Sequences are to be mentioned which are under the control of the promoters sequence express a fusion protein, where a component of the Fusion protein, the nucleic acid sequence to be expressed, the which one regulative protein sequence, such as. B. a signal or Transit peptide that delivers the gene product to the desired target site tet, is.

The production of an expression cassette according to the invention er follows, for example, by merging the myb11 promoter according to SEQ ID NO: 1 or a functional equivalent with one to expressing nucleotide sequence, optionally one for a Transit peptide coding sequence, preferably a chloropla star-specific transit peptide, which is preferably between the Promoter and the respective nucleotide sequence is arranged so such as a terminator or polyadenylation signal. Use for this one detects common recombination and cloning techniques like them for example in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labo ratory, Cold Spring Harbor, NY (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987) ben are.

The specificity of the inventive expression constructs and Vegetable, embryonic epidermis and flower vectors is particularly beneficial. The herbal epidermis poses so probably the primary barrier as well as the essential contact area of the plant organism to its environment. It has a important function in protecting the plant against biotic and ablotic stressors. Biotic stressors can be patho genes, browsing by animals, etc., ablotic stress can be Heat, cold, dryness, UV light etc. The epidermis has also a function in attracting beneficial insects by pigments storage or synthesis of volatile chemicals, it grants mechanical stability and prevents water loss. The Epi dermis also has an essential role in the seminal embryo in the absorption of water, gases and nutrients.

Often the natural defense mechanisms of the plant are part of it inadequate play against pathogens. The introduction of foreign genes The defense can come from plants, animals, or microbial sources strengthen. An example is the protection against insect caused damage in tobacco by expression of the Bacillus thuringiensis endotoxin under Kon trolls of the 35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37) or the protection of tobacco against fungal attack by means of Expres sion of a chitinase from the bean under the control of the CaMV Pro motors (Broglie et al., Science 1991, 254, 1194-1197).

Cold spells in the flowering period lead to significant each year Crop losses. A targeted expression of protective proteins ge targets in the flowering period can provide protection.

For such genetic engineering approaches to be highly efficient, one concentrated expression of the corresponding transgene generating nucleic acid sequence mainly in the outermost shell of the Plant, epidermis, or flower beneficial. A consti tutive expression throughout the plant can lead to the effect Example by diluting or questioning growth of the plant or the quality of the plant product gen. In addition, it can be ver strengthens to switch off the transgene ("gene silencing"). For this purpose promoters with specificity for the embryonic epider are needed mis and / or the bloom beneficial.

The person skilled in the art is familiar with a large number of proteins whose recombinant expression in the embryonic epidermis or the flower is advantageous. Furthermore, a number of genes are known to the person skilled in the art, by their repression or elimination by means of expression of a corresponding antisense RNA likewise advantageous effects can be achieved. Examples but not limiting of beneficial effects are: Achieving resistance to ablotic stress factors (heat, cold, dryness, increased moisture, environmental toxins, UV radiation) and biotic stress factors (pathogens, viruses, insects and diseases), which Improving food or feed properties, improving the growth rate or yield, achieving a longer or earlier flowering period, changing or enhancing the fragrance or color of the flowers. The nucleic acid sequences or polypeptides that can be used in these applications are to be mentioned as examples, but not by way of limitation:

• 1. Improved UV protection of the plant embryo through change change in pigmentation through the expression of certain polypeps tide like enzymes of flavonoid biosynthesis like the Chalconynt hase or the phenylalanine ammonium yase or certain enzymes DNA repair such as photolyase (Sakamoto A et al., DNA Seq. 1998; 9 (5-6): 335-40). Nucleus are particularly preferred acids necessary for the chalcone synthase from Arabidopsis thaliana (GenBank Acc.-No .: M20308), the 6-4 photolyase from Arabidop sis thaliana (GenBank Acc.-No .: BAB00748) or the blue light Photoreceptor / photolyase homolog (PHH1) from Arabidopsis thaliana (GenBank Acc.-No .: U62549) or functional equiva lente of the same code.
• 2. Improved protection of the plant embryo or flower against ablotic stress factors such as drought, heat, or Cold, for example, through overexpression of the "antifreeze" polypeptides from Myoxocephalus Scorpius (WO 00/00512), Myoxo cephalus octodecemspinosus, the Arabidopsis thaliana Trans script activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), a late embryogenic gene (LEA) for example from barley (WO 97/13843), calcium-dependent protein kinase gene nen (WO 98/26045), calcineurins (WO 99/05902), farnesyl transferases (WO 99/06580), Pei ZM et al., Science 1998, 282: 287-290), ferritin (Deak M et al., Nature Biotechnology 1999, 17: 192-196), oxalate oxidase (WO 99/04013; Dunwell JM Biotech nology and Genetic Engineering Reviews 1998, 15: 1-32), DREBIA factor (dehydration response element B 1A; Kasuga M et al., Nature Biotechnology 1999, 17: 276-286), genes from Manni tol or trehalose synthesis such as trehalose phosphate synthase or trehalose phosphate phosphatase (WO 97/42326), or by inhibiting genes such as trehalase (WO 97/50561). Particularly preferred are nucleic acids that are responsible for the trans ccriptional activator CBF1 from Arabidopsis thaliana (genetic Bank Acc.-No .: U77378) or the "antifreeze protein" from Myo xocephalus octodecemspinosus (GenBank Acc.-No .: AF306348) or code functional equivalents thereof.
• 3. Modification of the storage reserves, for example strength or Lipid modification, through the expression of certain enzymes to initiate fermentation processes after mashing of the seeds. The in seed grain clock different compartments of the food cherreserven in the seed and the enzymes to be expressed in the epidermis, which only goes through a process like mashing be brought into contact with each other. Examples are one improved mobilization of starch by expressing a Starch invertase from yeast or the modification of the lipid halted by expression of lipases, preferably of triacyl glycerol lipases. Nucleic acids are particularly preferred for the daily gene of the triacylglycerol lipase from Aspergillus oryzae (GenBank Acc.-No .: AB039325) or the SUC2 invertase from Saccharomyces cerevisiae (GenBank Acc.-No .: V01311) or code functional equivalents thereof.
• 4. Expression of metabolic enzymes for use in feed and the food sector, for example the expression of Phytase and cellulases. Nucleic acids are particularly preferred like the artificial code for a microbial phytase rende cDNA (GenBank Acc.-No .: A19451) or functional equi valents of the same.
• 5. Achievement of resistance to, for example, fungi, insects, Nematodes and diseases through targeted secretion or Accumulation of certain metabolites or proteins in the epi dermis of the embryo. Glucosinolates may be mentioned by way of example (Defense against nematodes), chitinases or glucanases and others Enzymes that destroy the cell wall of parasites, ribosome-in activating proteins (RIPs) and other plant proteins common resistance and stress reactions, such as those caused by injury or microbial infestation of plants or chemically by the Example induced salicylic acid, jasmonic acid or ethylene be, lysozymes from non-vegetable sources such as for the case play T4 lysozyme or lysozyme from different mammals, in secticidal proteins such as Bacillus thuringiensis endotoxin, α-amylase inhibitor or protease inhibitors (cowpea Trypsi ninhibitor), glucanases, lectins such as phytohemagglutinin, Snowdrop lectin, wheat germ agglutinin, RNAsen or Ribozymes. Particularly preferred are nucleic acids that are used for chit42 endochitinase from Trichoderma harzianum (GenBank Acc.- No .: S78423) or for the N-hydroxylating, multifunctional nelle cytochrome P-450 (CYP79) from Sorghum bicolor (GenBank Acc.-No .: U32624) or functional equivalents thereof encode.
• 6. Achieving insect repellancy or attraction, for example through increased release of volatile fragrances or messenger substances by for example enzymes of terpene biosynthesis.
• 7. Achieving a storage capacity in the epidermal cells that usually do not contain storage proteins or lipids with the aim of increasing the yield of these substances, for Example by expression of an acetyl-CoA carboxylase. Before Approved are nucleic acids that are responsible for acetyl-CoA carboxylase (Accase) from Medicago sativa (GenBank Acc.-No .: L25042) or code functional equivalents thereof.
• 8. Expression of transport proteins that support the uptake of Meta bolites, nutrients or water in the embryo and improve so the embryo growth, the metabolite composition or optimize the yield, for example by expressing a Amino acid transporter, which is responsible for the uptake of amino acids accelerated, or a monosaccharide transporter that the Promotes sugar intake. Preferred are preferred Nucleic acids necessary for the cationic amino acid transporter from Arabidopsis thaliana (GenBank Acc.-No .: X92657) or for the monosaccharide transporter from Arabidopsis thaliana (gene Bank Acc.-No .: AJ002399) or functional equivalents of the encode the same.
• 9. Expression of genes that cause an accumulation of fine chemicals lien, such as from tocopherols, tocotrienols or carotinides in effect the epidermis of the seed. An example is the Phy called toendesaturase. Preferred are nucleic acids that are used for the phytoene desaturase from Narcissus pseudonarcissus (GenBank Acc.-No .: X78815) or functional equivalents of the same ko date.
• 10. Modification of the wax ester formation or the composition the stored oligosaccharides to improve the protection zes against environmental influences or to improve digestion availability when used in feed or food. At the overexpression of the endoxyloglucan transfer may be playful called rase. Preferred are nucleic acids that are used for the Endo xyloglucan transferase (EXGT-A1) from Arabidopsis thaliana (gene Bank Acc.-No .: AF163819) or functional equivalents of the same encode ben.
• 11. Extending or canceling the resting phase by expressing Transcription factors involved in the regulation of dormancy-re intervening in levant genes. The overexpression is an example sion of the ABI3 protein (abscisic acid insensitive gene) or des Viviparous-1 protein (VP-1). The germination time changes Modification can result in an increase in yield and a shortened embryo nal development. Preferred are nucleic acids that for the Viviparous-1 protein from Zea mays (GenBank Acc.-No .: M60214) or the ABI3 protein (abscisic acid insensitive gene) from Arabidopsis thaliana (GenBank Acc.-No .: X68141) or code functional equivalents thereof.
• 12. Production of sterile plants by preventing the loading fruiting and / or germination with the help of the expression of a suitable inhibitor for example a toxin in flowers and in the seed.
• 13. Production of neutraceuticals such as polyunsaturated saturated fatty acids such as arachidonic acid or EP (Eicosapentaenoic acid) or DHA (docosahexaenoic acid) Expression of fatty acid elongases and / or desaturases or Production of proteins with improved nutritional value such as for example with a high proportion of essential amino acids ren (e.g. the methionine-rich albumin of the brazil nut). Nucleic acids which are preferred for the methionine-rich 25 Albumin from Bertholletia excelsa (GenBank Acc.-No .: AB044391), the Δ6-acyl lipid desaturase from Physcomitrella patens (GenBank Acc. No .: AJ222980; Girke et al 1998, The Plant Journal 15: 39-48), the Δ6-desaturase from Mortierella alpina (Sakura dani et al 1999 Gene 238: 445-453), the Δ5-desaturase from Cae norhabditis elegans (Michaelson et al. 1998, FEBS Letters 439: 215-218), the Δ5-fatty acid desaturase (des-5) from Caenor habditis elegans (GenBank Acc.-No .: AF078796), the Δ5-Desatu Lawn from Mortierella alpina (Michaelson et al. JBC 273: 19055-19059), the Δ6 elongase from Caenorhabditis elegans (Beau doin et al. 2000, PNAS 97: 6421-6426), the Δ6 elongases Physcomitrella patens (Zank et al. 2000, Biochemical Society Transactions 28: 654-657) or functional equivalents of the encode the same.
• 14. Production of pharmaceuticals such as antibodies or vaccines as described by Hood EE, Jilka JM. Curr Opin Biotechnol. 1999 Aug; 10 (4): 382-6; Ma JK, Vine ND. Curr Top Microbiol Immunol. 1999; 236: 275-92.
• 15. Modification of the fiber content in foods based on seeds sis by, for example, expressing antisense transcripts caffeinic acid O-methyltransferase or cinnamoyl alcohol holdehydrogenase gene.
• 16. Inhibition of maturation of the seed coat by expression of for example ribonuclease genes or transcription factors to achieve an increased seed size, to reduce the relative biomass of the seed coat and to facilitate the Revelation of the seed. An overexpression is an example here sion of the ANT (AINTEGUMENTA) gene. Nu are preferred small acids that are responsible for the AINTEGUMENTA gene from Arabidopsis thaliana (GenBank Acc.-No .: U40256) or functional equiva encode lente of the same.

Further examples of advantageous genes are mentioned, for example at Dunwell JM, Transgenic approaches to crop improvement, J Exp Bot. 2000; 51 Spec No; Page 487-96.

Functional equivalents here means - analogous to the definition the functional equivalents of the promoter with specificity for the embryonic epidermis and / or the flower - all the sequences which have essentially the same function d. H. to the Function (for example a substrate conversion or a signal transduction) are capable as well as the example mentioned Protein. The functional equivalent may be in others Distinct features are quite different. For example, it can be a have here or lower activity or via further radio functionalities.

Functional equivalents also means sequences that for Fusion proteins consisting of one of the preferred proteins and other proteins, for example another preferred protein or also encode a signal peptide sequence.

The person skilled in the art is also known to have the above-described Genes not directly using the genes coding for these genes Express nucleic acid sequences or, for example, by anti sense must repress. It can also be artificial, for example Use transcription factors of the zinc finger protein type (Beerli RR et al., Proc Natl Acad Sci USA 2000; 97 (4): 1495-500). These factors are stored in the regulatory areas of the endogenous genes to be expressed or repressed on and cause, depending on the design of the factor, an expression or Repression of the endogenous gene.

Another object of the invention relates to transgenic organisms men, transformed with at least one Ex according to the invention compression cassette or a vector according to the invention, as well as Cells, cell cultures, tissues, parts - such as at plant organisms leaves, roots, etc. - or reproductive well derived from such organisms.

Organism, parent or host organisms become prokaryo table or eukaryotic organisms such as microor understood organisms or plant organisms. Preferred Wed Microorganisms are bacteria, yeasts, algae or fungi.

Preferred bacteria are bacteria of the genus Escherichia, Er winia, Agrobacterium, Flavobacterium, Alcaligenes or Cyanobak for example of the genus Synechocystis.

Above all, microorganisms which cause infection are preferred of plants and thus for the transfer of the invention Constructs are enabled. Preferred microorganisms are those from the genus Agrobacterium and in particular the species Agrobacte rium tumefaciens.

Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.

Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neu rospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, table 6 be wrote mushrooms.

Host or starting organis preferred as transgenic organisms Men are above all plants. Are included under the Invention of all genera and species of higher and lower plant zen of the plant kingdom. The tires are also included Plants, seeds, sprouts and seedlings, as well as derived therefrom killed parts, reproductive material and cultures, for example cell culture Ren. Mature plants means plants of any development phase beyond the seedling. Seedling means a young one, immature plant at an early stage of development.

Annual, perennial, monocotyledon and dicotyledon plants are preferred host organisms for the production of transgenic Plants. Expression of genes in the embryonic epidermis and especially the flower is also advantageous in all jewelry plants, useful or ornamental trees, flowers, cut flowers, bouquets chern or lawn. Exemplary but not restrictive are too call angiosperms, bryophytes such as Hepaticae (Liverwort) and musci (moss); Pteridophytes like ferns, Horsetail and lycopod; Gymnosperms such as conifers, cycads, Ginkgo and Gnetalen; Algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (Diatoms) and Euglenophyceae.

Plants within the scope of the invention include and do not include by way of example restrictive the families of Rosaceae like Rose, Ericaceae like Rhododendrons and azaleas, Euphorbiaceae such as poinsettias and Croton, Caryophyllaceae such as carnations, Solanaceae such as petunias, Gesneriaceae like the African violet, Balsaminaceae like that Balsam, Orchidaceae like orchids, Iridaceae like gladioli, Iris, freesia and crocus, compositae such as marigold, Geraniaceae like geraniums, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others more.

Exemplary but not restrictive for flowering plants are too name the families of the Leguminosae such as pea, alfalfa and soy; Gramineae such as rice, corn, wheat; Solanaceae like tobacco and others more; the Umbelliferae family, particularly the Daucus genus (especially the kind carota (carrot)) and Apium (very special the other kind graveolens dulce (Selarie)) and others more; the company milie of the Solanacea, especially the genus Lycopersicon, quite be especially the species esculentum (tomato) and the genus Solanum, whole especially the species tuberosum (potato) and melongena (eggplant) and others more; and the genus Capsicum, especially the species annum (pepper) and others more; the Leguminosae family, be especially the genus Glycine, especially the species max (soy bean) and others more; and the family of the Cruciferae, especially the genus Brassica, especially the species campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and the genus Arabidopsis, especially the species thaliana and others; the family of Compositae, especially the genus Lactuca, especially the species Sativa (salad) and others more.

The transgenic plants according to the invention are selected in particular from among monocotyledonous crop plants, such as, for example, types of grain such as wheat, barley, millet, rye, triticale, maize, rice or oats and sugar cane. Furthermore, the transgenic plants according to the invention are in particular selected from dikotylene crop plants, such as, for example
Brassicacae such as rapeseed, cress, Arabidopsis, cabbage species or canola, leguminosae such as soy, alfalfa, peas, alfalfa, beans or peanuts
Solanaceae such as potato, tobacco, tomato, aubergine or paprika, Asteraceae such as sunflower, marigold, lettuce or calendula, Cucurbitaceae such as melon, pumpkin or zucchini,
as well as flax, cotton, hemp, flax, red pepper, carrots, carrots, sugar beet and the various tree, nut and wine species.

Arabidopsis thaliana, Nicotiana tabacum, Tagetes erecta, Calendula officinalis and all genera and Species used as food or feed, like the types of grain described, or used for production suitable for oils, such as oilseeds (such as rapeseed), types of nuts, Soy, sunflower, pumpkin and peanut.

Plant organisms for the purposes of the invention are also white more photosynthetically active, capable organisms, such as play algae or cyanobacteria, as well as mosses.

Preferred algae are green algae, such as, for example, algae Genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.

The production of a transformed organism or a transformed cell requires that the appropriate DNA in the corresponding host cell is introduced. For this operation, which is referred to as transformation stands a multitude of Methods are available (see also Keown et al. 1990 Methods in Enzymology 185: 527-537). In this way, the DNA can go straight through Microinjection or bombardment with DNA-coated Microparticles are introduced. The cell can also chemically for example with polyethylene glycol, be permeabilized, so that the DNA can get into the cell by diffusion. The DNA can also be obtained by protoplast fusion with other DNA-containing Units such as minicells, cells, lysosomes or liposomes succeed gen. Electroporation is another suitable method for a guidance of DNA, in which the cells are reversible by an elec tric impulse be permeabilized.

In the case of plants, the methods described for trans formation and regeneration of plants from plant tissues or Plant cells for transient or stable transformation ge uses. Suitable methods are, above all, the protoplast trans formation by polyethylene glycol-induced DNA uptake, the biolistic processes with the gene gun, the so-called particle bombardment method, electroporation, incubation dry embryos in a solution containing DNA and the microinjection.

In addition to these "direct" transformation techniques, a trans formation also through bacterial infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti or Ri plasmid), which on the plant is transmitted after Agrobaterium infection. A part this plasmid, called T-DNA (transferred DNA), is in the Ge integrated into the plant cell.

The Agrobacterium-mediated transformation is best for dicotyledonous, diploid plant cells, whereas the direct transformation techniques are suitable for any cell type.

The introduction of an expression cassette according to the invention in Cells, preferably in plant cells, can be beneficial under Use of vectors can be realized. Vectors can be used at playful plasmids, cosmids, phages, viruses or agrobacts be rien.

In an advantageous embodiment, the introduction of the Expression cassette realized by means of plasmid vectors. Preferred are those vectors that allow stable integration of the expresses Enable sion cassette in the host genome.

In the case of injection or electroporation of DNA in vegetable che cells are no special requirements for the used Plasmid provided. Simple plasmids such as those of the pUC series can be used. Should complete plants from the transfor mated cells are regenerated, it is necessary that there is an additional selectable marker gene on the plasmid is located.

Transformation techniques are available for different monocots and dicotyledonous plant organisms described. Furthermore, ver different possible plasmid vectors for introducing foreign ones Genes are available in plants that are usually replicas origin for a reproduction in E. coli and a marker gene for contain a selection of transformed bacteria. Examples are pBR322, pUC series, M13mp series, pACYC184 etc.

The expression cassette can be inserted into the vector via a suitable Restriction cleavage sites are introduced. The resulting Plasmid is first introduced into E. coli. Correctly transfor mated E.coli are selected, bred and the recombi nante plasmid obtained using methods familiar to those skilled in the art.

Restriction analysis and sequencing can be used to identify the Check cloning step.

Transformed cells d. H. those that intertwine the introduced DNA contained in the host cell's DNA, can be of untrans formed are selected if a selectable Mar not part of the introduced DNA. As a marker, playful every gene act that has antibiotic resistance capable of imparting tics or herbicides. Transformed cells, expressing such a marker gene are able to in the Presence of concentrations of an appropriate antibiotic or herbicide to survive that is an untransformed wild type kill. Example are the bar gene that resistance to the Her Bicidal Phosphinotricin confers (Rathore KS et al., Plant Mol Biol. 1993 Mar; 21 (5): 871-884), the nptll gene that resistance to Kanamycin, the hpt gene, confers resistance to hygromycin confers, or the EPSP gene, which gives resistance to the herbicide Glyphosate confers.

Depending on the method of DNA introduction, additional genes can be found on the Vector plasmid may be required. If Agrobacteria are used, the expression cassette can be integrated into special plasmids ren, either in an intermediate vector (English: shuttle or in termediate vector) or a binary vector. If for example a Ti or Ri plasmid is to be used for transformation, is at least the right limit, but mostly the right one and the left border of the Ti or Ri plasmid T-DNA as a flan kating region with the expression cassette to be introduced ver bound. Binary vectors are preferably used. Binary vector Ren can replicate in E. coli as well as in Agrobacterium ren. They usually contain a selection marker gene and a Left or polylinkers flanked by the right and left T-DNA Delimiting sequence. You can directly in Agrobacterium transfor (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The selection marker gene allows selection trans formed agrobacteria and is, for example, the nptll gene, the gives resistance to kanamycin. In this case as Host organism acting Agrobacterium should already be a Plasmid with the vir region included. This is for the transmission The transfer of the T-DNA to the plant cell is required. Such a transformed Agrobacterium can be used to transform plants cher cells can be used.

The use of T-DNA to transform plant cells has been intensively investigated and described (EP 120516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4: 1-46 and An et al. (1985) EMBO J. 4: 277-287). Different binary vectors are known and some are commercially available such as pBIN19 (Clontech Laboratories, Inc. U.S.A.).

For the transfer of the DNA to the plant cell, plants are used Liche explants with Agz-obacterium tumefaciens or Agrobacterium rhizogenes co-cultivated, starting from infected plant matter rial (e.g. leaf, root or stem parts, but also protopla most or suspensions of plant cells) can whole plants using a suitable medium such as An tibiotics or biocides for the selection of transformed cells ent can hold, can be regenerated. The plants obtained can then on the presence of the imported DNA, here the invention expression cassette must be screened. Once the DNA integrated into the host genome is the corresponding genotype usually stable and the corresponding insertion is also in found again in the following generations. Usually contains the integrated expression cassette a selection marker that the transformed plant has a resistance to a biocide (for Example a hericide) or an antibiotic such as kanamycin, G 418, Bleomycin, hygromycin or phosphinotricin etc. confers. The Se Lesson marker allows the selection of transformed cells of untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84). The plants obtained can be used in a customary manner bred and crossed. Should two or more generations ten cultivated to ensure that the genomic Integration is stable and inheritable.

The methods mentioned are, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42: 205-225 (1991) be wrote. The construct to be expressed is preferably in cloned a vector suitable for Agrobacterium tumefa ciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12: 8711 (1984)).

Once a transformed plant cell has been created, can a complete plant using be known procedures can be obtained. One goes here by way of example from callus cultures. From these still undifferentiated cells The formation of shoots and roots can be achieved in a known manner be induced. The sprouts obtained can be planted out and be bred.

The efficiency of the expression of the transgenically expressed nucleus acids can, for example, increase in vitro by means of shoot meristem tion using one of the selection measurements described above methods are determined.

According to the invention are also of the above-described transge nen organisms derived cells, cell cultures, parts - such as for Example with transgenic plant organisms roots, leaves etc. -, and transgenic reproductive material such as seeds or fruits.

According to the invention which can be consumed by humans and animals, genetically Modified plants can also, for example, directly or after processing known per se as food or feed tel can be used.

Another object of the invention relates to the use of above-described transgenic organisms according to the invention and the cells derived from them, cell cultures, parts - how to Example with transgenic plant organisms roots, leaves etc. -, and transgenic reproductive material such as seeds or fruits, for the production of food or animal feed, pharmaceuticals or fine chemicals.

A method for recombinant production is also preferred of pharmaceuticals or fine chemicals in host organisms being a host organism with one of the expressions described above Cassette is transformed and this expression cassette or contains more structural genes necessary for the desired fine Code chemical or the biosynthesis of the desired fineche catalyze the transformed host organism and the desired fine chemical from the breeding me dium is isolated. This procedure is for fine chemicals like Enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavors, aromas and colors can be used widely. The production of tocopherols and toco is particularly preferred trienols and carotenoids. The breeding of the transformed Host organisms as well as isolation from the host organisms or from the cultivation medium is carried out using ver known to the person skilled in the art travel. The production of pharmaceuticals, such as anti bodies or vaccines are described in Hood EE, Jilka JM. Curr Opin Biotechnol. 1999 Aug; 10 (4): 382-6; Ma JK, Vine ND. Curr Top Microbiol Immunol. 1999; 236: 275-92.

Sequences

• 1. SEQ ID NO: 1 promoter and 5 'untranslated region of the macaw bidopsis thaliana myb11 gene.
• 2. SEQ ID NO: 2 203Sbp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene.
• 3. SEQ ID NO: 3 oligonucleotide primers, oligo- (dT) primers
• 4. SEQ ID NO: 4 oligonucleotide primers
• 5. SEQ ID NO: 5 oligonucleotide primers
• 6. SEQ ID NO: 6 oligonucleotide primers
• 7. SEQ ID NO: 7 oligonucleotide primers
• 8. SEQ ID NO: 8 oligonucleotide primers, forward primer Z17F2
• 9. SEQ ID NO: 9 oligonucleotide primer, reverse primer Z17R3
• 10. SEQ ID NO: 10 oligonucleotide primers, PCR adapter primer 1
• 11. SEQ ID NO: 11 oligonucleotide primers, myb11 forward primers
• 12. SEQ ID NO: 12 oligonucleotide primer, myb11 reverse primer
• 13. SEQ ID NO: 13 oligonucleotide primers
• 14. SEQ ID NO: 14 oligonucleotide primers
• 15. SEQ ID NO: 15 888bp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene.
• 16. SEQ ID NO: 16 282bp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene
• 17. SEQ ID NO: 17 1394bp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene
• 18. SEQ ID NO: 18 1191bp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene
• 19. SEQ ID NO: 19 665 bp fragment from promoter and 5'-untransla the region of the Arabidopsis thaliana myb11 gene

Illustrations

The following figures show in-situ hybridizations of different plant organs at different times of development:

• 1. Fig. 1 (I and II) show a section through a seed and thus through the plant embryo (Arabidopsis tha liana). "in" denotes the integument, "h" the hypocotyl, "c" the cotyledon and "r" the root. Fig. 1 (I) shows the section without showing the expression region. Fig. 1 (II) illustrates the expression region of the myb11 promoter, which runs along the white, dashed line (marked by the white arrow). This region corresponds to the embryonic epidermis.
• 2. Fig. 2 (I and II) show a section through the plant flower bud (Arabidopsis thaliana). "sp" denotes the stigmatic papilla, "w" the ovarian wall, "st" the stigma, "pl" the placentae (nutrient tissue) and "ov" the egg. Fig. 2 (I) shows the section without showing the expression region. Fig. 2 (II) illustrates the Expres sion region of the myb11 promoter, which can be found strongly in the hatched regions, less strongly in the dotted regions. These regions correspond on the one hand to the ovarian wall and on the other hand to the ovaries themselves.
• 3. Fig. 3 (I and II) show a section through the Ovarienanla gene of Arabidopsis thaliana. "al" denotes the pollen sac (English: anther locules), "g" the gynoecium, "op" the egg lay (English: ovule primordia) and "sp" a petal (sepale). Fig. 3 (I) shows the section without showing the expression region. Fig. 3 (II) illustrates the expression region of the myb11 promoter, which can be found in the white-hatched regions (marked by white arrows). These regions correspond to the egg enclosures.
• 4. Fig. 4 (I and II) show a section through the plant flower systems (Arabidopsis thaliana). "im" denotes the flower meristem (tunica). The numbers 2 and 6 describe flowers in the flower development stage 2 or 6 (Arabidopsis At las of Morphology, Bowman J ed., Springer Verlag, New York, USA, 1995). Fig. 4 (II) shows the section without illustrating the expression region. Fig. 4 (I) illustrates the Expres sion region of the myb11 promoter, which can be found in the white-hatched regions. These regions correspond to the flower meristem and the flower tip (development stage 2) or the stamen and the petal primordia (development stage 6).
• 5. Fig. 5 (I) shows a schematic representation of the expression in flower stage 2. "fa" denotes the flower apex. The expression region of the mybll promoter can be found in the hatched region. Fig. 5 (II) shows a schematic representation of the expression in flower stage 6. "p" denotes the petal primordium, "ms" denotes the stamen), "g" the gynoecium. The expression region of the myb11 promoter can be found in the hatched region
• 6. Fig. 6: The result of an RT-PCR using various tissues to determine the expression pattern of the mybll promoter is shown. Expression can be detected in the seedling (day 4), the flower bud, flower and wilting flower, as well as the green pods.
• 7. Fig. 7: Schematic representation of the vectors pCR-TOPO-myb11 (I), pGPTV-myb11 :: GUS (II), pGPTV1-myb11 :: GUS (III) and pGPTV2-myb11 :: GUS (IV). The direction of the arrow indicates the orientation of the promoter, with the arrowhead pointing in the direction of the start codon. The abbreviations have the following meanings:
  myb11: promoter or promoter fragment of the myb11 promoter
  NOS-T: terminator sequence of nopaline synthase (NOS)
  GUS: reporter gene (bacterial β-glucuronidase)
• 8. Fig. 8: Schematic representation of the vectors pGPTV3-myb11 :: GUS (V), pGPTV4-myb11 :: GUS (VI), pGPTVS-myb11 :: GUS (VII) and pGPTV6-myb11 :: GUS (VIII). The direction of the arrow indicates the orientation of the promoter, the arrowhead pointing in the direction of the start codon. The abbreviations have the following meanings:
  myb11: promoter or promoter fragment of the myb11 promoter
  NOS-T: terminator sequence of nopaline synthase (NOS)
  GUS: reporter gene (bacterial β-glucuronidase)
• 9. Fig. 9: Schematic representation of the vectors pBSK-myb11 (IX) and pBM-myb11 (X). The direction of the arrow indicates the orientation of the promoter, the arrowhead pointing in the direction of the start codon. The abbreviations have the following meanings:
  myb11: promoter or promoter fragment of the myb11 promoter

Examples General methods

The chemical synthesis of oligonucleotides can, for example, in a known way, according to the phosphoamidite method (Voet, Voet, 2. Edition, Wiley Press New York, pages 896-897). The in Cloning carried out within the scope of the present invention steps such as B. restriction digests, agarose gel electrophoresis rese, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, linking DNA frag ments, transformation of E. coli cells, cultivation of bacteria, Propagation of phages and sequence analysis of recombinant DNA as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6 described carried out. The sequences The treatment of recombinant DNA molecules is carried out with a laser fluo Rescence DNA sequencer from ABI according to the method of San ger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).

example 1 Plant growth conditions for tissue-specific cal RT-PCR analysis

To get 4 or 7 day old seedlings, unge about 400 seeds (Arabidopsis thaliana ecotype Columbia) on the surface sterilized with an 80% ethanol solution for 2 minutes, with treated with a sodium hypochlorite solution (0.5% v / v) for 5 minutes, washed three times with distilled water and at 4 ° C for 4 days incubated to ensure uniform germination. The seeds are then placed on Petri dishes with MS medium (Sigma M5519) with the addition of 1% sucrose, 0.5 g / l MES (Sigma M8652), 0.8% Difco-BactoAgar (Difco 0140-01), pH 5.7 incubated. The seedlings are in a 16-hour light / 8-hour light Dark cycle (Philips 58W / 33 white light lamps) at 22 ° C grown and after 4 or 7 days after the start of the germination phase harvested.

To obtain roots, 100 seeds are used as described above ben sterilized, incubated at 4 ° C for 4 days and then in 250 ml fla with MS medium (Sigma M5519) with the addition of a further 3% Sucrose and 0.5 g / l MES (Sigma M8652), pH 5.7. the Seedlings are in a 16 hour light / 8 hour dun cold cycle (Philips 58 W / 33 white light lamps) at 22 ° C, Grown at 120 rpm and harvested after 3 weeks. For all others The plant organs used are the seeds on unit earth (Type VM, Manna-Italia, Via S. Giacomo 42, 39050 San Giacomo / Laives, Bolzano, Italy) sown, incubated for 4 days at 4 ° C to ensure even germination and then in one 16-hour light / 8-hour dark cycle (OSRAM Lumi lux Daylight 36 W / 12 fluorescent tubes) grown at 22 ° C. Young Rosette leaves are picked at the 8-leaf stage (after 3 weeks) tet, ripe rosette leaves become shortly before the after 8 weeks Stalk formation harvested. Inflorescences (apices) of the discharging Stalks are harvested shortly after imposition. Stem, stem Gel leaves and flower buds are in development stage 12 (Bowmann J (ed.), Arabidopsis, Atlas of Morphology, Springer New York, 1995) harvested before the stamen develop. Opened Flowers appear in stage 14 immediately after the stamen develop harvested. Wilting flowers are harvested at stages 15 to 16. The green and yellow pods used had a length of 10 to 13 mm.

Example 2 RNA extraction and RT-PCR analysis

Total RNA is isolated from the organs of the plant described in Example 1 at various times of development as described (Prescott A, Martin C Plant Mol Biol Rep 1987, 4: 219-224). The reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect the Atmybll gene transcript. All RNA samples are treated with DNaseI (15 units, Boehringer, Mannheim) before cDNA synthesis. The first strand cDNA synthesis is carried out starting from 6 µg total RNA with an oligo (dT) primer and RT Superscript ^™ II enzyme (300 units) according to the manufacturer's instructions in a total volume of 20 µl (Life Technologies, Gaithersburg, MD). 150 ng oligo- (dT) are added to the RNA in a final volume of 12 μl. The batch is heated at 70 ° C. for 10 min and then immediately cooled on ice. Then 4 μl of the 5X first strand buffer, 2 μl 0.1M DTT, 1 μl 10 mM dNTP mix (each 10 mM dATP, dCTP, dGTP and dTTP) and RNase inhibitor (5 units, Boehringer Mannheim) are added. The batch is heated to 42 ° C. for 2 min, RT Superscript ™ enzyme (300 units, Life Technologies) is added and incubated at 42 ° C. for 50 min. A 35-base long oligonucleotide with 17 dT residues and an adapter sequence is used as the oligo (dT) primer (Frohman MA (1990) Academic Press, San Diego, CA. pp. 28-38):

5'-GGGAATTCGTCGACAAGC (T) ₁₇ -3 ') (SEQ ID NO: 3)

The first strand cDNA is used as a template for the PCR. The following primers are used for the PCR:

Forward primer Z17F2: 5'-GCCAATACCGTCGAGAATGCGCC-3 '(SEQ ID No: 8)

Reverse primer Z17R3: 5'-TCGTCAATATCCAACGGTTCTCC-3 '(SEQ ID NO: 9)

The PCR reaction mixture is composed as follows:
2.5 µg first strand cDNA
1X PCR Buffer II Perkin Eimer (Warrington, UK)
2.5 mM mM MgCl ₂
200 µM each of dATP, dCTP, dGTP and dTTP
0.5 µM of each of the oligonucleotide primers Z127F2 and Z17F3 (SEQ ID No .: 8 and 9)
2.5 Units AmpliTaq (Perkin Bucket)
least. Water up to a final volume of 50 µl.

The following temperature program is used (PTC-100TM Model 96V; MJ Research, Inc., Watertown, Massachussetts):
1 cycle with 150 sec at 94 ° C
20 cycles of 94 ° C for 45 sec, 55 ° C for 1 min and 72 ° C for 2 min.
1 cycle at 72 ° C for 7 min

To ensure linearity of the amplification process, only 20 PCR cycles are carried out. The PCR products are separated by means of 2% w / v agarose gel electrophoresis, blotted onto a Hybond N + nylon membrane (Amersham, England) and hybridized with fluorescein-labeled probes. The production of the probes and the detection reaction take place as indicated in the manufacturer's report from Amersham. The standardization of the method is verified by using primers specific for the Arabidopsis ACT1 gene, which codes for actin (An YQ et al. Plant Cell 1996, 8: 15-30). The following primers are used for the mRNA detection of At MYB11:

Forward primer Z17F2: 5'-GCCAATACCGTCGAGAATGCGCC-3 '(SEQ ID NO: 8)

Reverse primer Z17R3: 5'-TCGTCAATATCCAACGGTTCTCC-3 '(SEQ ID NO: 9)

The size of the amplified PCR product is 562 bp. The PCR reaction is only carried out over 25 cycles under the conditions mentioned above. (EMBL AF062863; Kranz et al., 1998). The result of the RT-PCR analysis is shown in FIG. 6.

Example 3 Analysis of Atmyb11 expression by in situ hybridi ization

Flowers and pods are collected at different stages of development from 7-week-old plants maintained in soil as described above. The samples are immediately fixed in freshly prepared 4% p-formaldehyde solution in PBS buffer (130 mM NaCl, 7 mM Na ₂ HPO ₄ , 3 mM NaH ₂ PO ₄ ) under vacuum for 3 hours. The fixed material is stored in 70% ethanol at 4 ° C until further use. Fixation and embedding work are carried out according to the instructions of Dolfini et al. (Dolfini S et al., Dev. Genet. 1992, 13, 264-276) with the modification that each step described was shortened to a duration of 1 hour. The template for the sample for in-situ hybridization is produced as described in the instructions for the Lig'nScribe ^™ PCR Promoter Addition Kit (Ambion, USA). For this purpose, 20 ng of the PCR fragment obtained with the primers Z17F2 and Z17R3 starting from a partial cDNA clone of the AtMYB11 gene (EMBL AF062863; Kranz et al., 1998 (see above), counter to its reading direction, behind a T7 promoter adapter cloned (Ambion, USA). This ligation reaction contains 1 µl of the 10X ligation buffer, 1 µl of the promoter adapter, 1 µl of T4 DNA ligase and distilled water in a total volume of 10 µl. The ligation reaction is incubated for 15 min at room temperature. The template for the The antisense strand of the AtMYB11 probe is carried out by means of PCR using part of the ligation reaction and the primers Z17F2 (SEQ ID NO: 8) and Ambion PCR primer 1. For this purpose, 2 μl of the ligation reaction and the primers Z17F2 and the PCR adapter primer 1 are added

PCR adapter primer 1: 5'-GCTTCCGGCTCGTATGTTGTGTGG-3 '(SEQ ID NO: 10)

mixed. The primers Z17R3 and PCR adapter primer 1 are used to produce the template for the sense strand of the AtMYB11 probe. The PCR conditions for producing the templates for the antisense and sense strands of the AtMYB11 probes are identical and are carried out under the conditions described in Example 2. Deviating from this, 35 cycles are carried out and only a concentration of 0.25 mM of the primers used is used. Sense and antisense DIG-11-UTP labeled RNA probes are synthesized using T7 RNA polymerase with reagents from Roche Diagnostics: The following components are mixed in the specified order in an RNase-free Eppendorf tube, which is kept on ice. The final concentration in each case is given in a reaction volume of 20 μl. 0.1 µg ethanol-precipitated PCR product, 1 × labeling mixture (as 10 × mixture with 10 mM ATP, 10 mM CTP, 10 mM GTP; 6.5 mM UTP, 3.5 mM DIG-UTP in Tris-HCl, pH 7.5 ), 1 × transcription buffer (as 10 × buffer with 400 mM Tris-HCl, pH 8.0, 60 mM MgCl ₂ , 100 mM dithioerythritol, 20 mM spermidine, 100 mM NaCl, 1 unit / ml RNase inhibitor) and 40 units of T7 RNA polymerase. The mixture is shaken briefly and mixed and centrifuged for a few seconds. The mixture is then incubated at 37 ° C. for at least 2 hours. To digest the DNA-PCR product, 20 units of DNase are added and incubated for 15 min at 37 ° C. To terminate the reaction, 2 µl of the supplied EDTA solution are added.

8 µm thick sections of the fixed plant material are cut through a microtome using S35 type microtome blades (Fea ther, Japan) cut (Minot microtome type 1212; Leitz Wetzlar, Germany), applied to slides loaded with poly-L-lysine and treated as in Coen et al. described (Coen Es et al., Cell 1990, 63: 1311-1322). Before hybridization, the Sections inserted in xylene to remove the paraffin and on finally rehydrated with a series of ethanol dilutions sated. This is followed by an incubation with 1 μg / ml proteinase K for 30 min at 37 ° C in 100 mM Tris-HCl (pH 8), 50 mM EDTA. Afterward is acetylated with acetic anhydride, with a dilution series of Ethanol dehydrated and air dried. The DIG-11-UTP labeled RNA probes undergo mild alkaline hydrolysis with heating at 60 ° C for one hour in 100 mM carbonate buffer (pH 10.2) exposed to an average fragment length of reaching about 150 bases.

Approximately 20 ng each of the DIG-11-UTP labeled RNA probe in 50 µl hybridization buffer preheated to 70 ° C (50% formamide, 300 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1X Denhardts, 10 % Dextran sulfate, 10 mM DTT, 250 ng / ml tRNA, 100 μg / ml polyA) are used for each slide and incubated with the tissue sample at 45 ° C. overnight. The supports are 45 min in 2X SSC (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0) at room temperature, 45 min in 2X SSC at 55 ° C, 45 min in 0.5X SSC at 55 ° C and then treated with 20 μg / ml RNase A in NTE (0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA) at 37 ° C. for 30 min. The supports are then washed again for 15 minutes in 2X SSC at room temperature and for 15 minutes in 2X SSC at 50.degree. The immunological detection of the probe is carried out according to the instructions of the "digoxige nin-nucleic acid detection kit" (Roche Diagnostics): For this purpose, the carriers are gently agitated for 1 hour in 1% blocking solution (Roche Diagnostics) in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl and then incubated for 30 min in buffer A (0.5% bovine serum albumin, 0.3% Triton-X100, 100 mM Tris-HCl (pH 7.5), 150 mM NaCl). The carriers with the antibody conjugate are then incubated in a 1 12612 00070 552 001000280000000200012000285911250100040 0002010053519 00004 12493: 1500 dilution in buffer A and washed three times for 20 min in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl. The carriers are then washed for 5 min in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl ₂ and for 48 hours in a solution of 0.34 mg / ml nitro blue tetrazolium salt and 0.175 mg / ml 5-bromo- 4-chloro-3-indolylphosphate toluidinium salt incubated in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl ₂ . The color reaction is stopped with 10 mM Tris-HCl (pH 8), 1 mM EDTA and the sections are washed with gentle shaking in 95% ethanol, then with an ethanol dilution series and finally with sterile water before they are consolidated with Euparal (BDH) will. The tissue is then documented with a camera under a suitable microscope (e.g. Leica DMRB).

Example 4 Cloning of the AtMYB11 promoter

In order to isolate the complete AtMYBII promoter, genomic DNA is extracted from Arabidopsis thaliana (Columbia ecotype) as described (Galbiati M et al. Funct. Integr Genomics 2000, 1: 25-34). The isolated DNA is used as template DNA in a PCR using the following primers:

Forward primer: 5'-AAGCTTTTGATTTTACAATGAGG-3 '(SEQ ID NO: 11)

Reverse primer: 5'-CCCGGGAAAATCACTCACTTCACT-3 '(SEQ ID NO: 12)

The amplification is carried out as follows:
80 ng of genomic DNA
1X Expand ^TM Long Template PCR Buffer 1
2.5 mM MgCl ₂ ,
350 µM each of dATP, dCTP, dGTP and dTTP
300 nM each of each primer (SEQ ID NO: 11 and 12)
2.5 units of Expand ™ Long Template Polymerase (Roche Diagnostics).
in a final volume of 25 µl.

The following temperature program is used (PTC-100TM Model 96V; MJ Research, Inc., Watertown, Massachussetts):
1 cycle with 120 sec at 94 ° C
35 cycles with 94 ° C for 10 sec, 55 ° C for 30 sec and 68 ° C for 3 min.
1 cycle at 68 ° C for 30 min

The PCR product is cloned into the vector pCR4-TOPO (Invitrogen) according to the manufacturer's instructions, ie the PCR product obtained is inserted into a vector with T overhangs by means of its A overhangs and a topoisomerase. The construct obtained is pCR4-TOPO-myb11 ( Fig. 7, I).

Example 5 Deletion analysis

The method is from Rouster (Rouster J et al., Plant Journal 11 (3): 513-23, 1997) and Sambrook (Sambrook et al., Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, 1989).

A fine mapping of the myb11 promoter, i. H. a narrowing of the nucleic acid sections relevant to its specificity by producing various reporter gene expression vectors, on the one hand the entire promoter area, on the other hand different contain fragments of the same. On the one hand, the entire promoter region or fragments thereof in the pGPTV-GUS-Kan Vector (Becker D et al., 1992 Plant Mol Biol 20: 1195-1197). There for on the one hand fragments are used, which by the use Creation of restriction enzymes for the internal restriction interfaces in the full-length promoter sequence can be obtained. On the other hand, PCR fragments are used which are identified by Pri mer introduced interfaces are provided.

To clone the full-length promoter, the vector pCR4-TOPO-myb11 is digested with HindIII and SmaI and the fragment isolated from a 1% agarose gel is cloned into the pGPTV-GUS channel. The construct obtained is referred to as pGPTV-myb11 :: GUS ( Fig. 7, construct II).

For a first shortened promoter, pCR4-TOPO-myb11 is digested with EcoRI and the approximately 2050 base pair fragment isolated from a 1% agarose gel is subcloned into pBluescript SK (Stratagene). The insert is obtained from the resulting vector pBSK myb11 ( FIG. 9, construct IX) by digestion with HindIII and SmaI, isolated on a 1% agarose gel and cloned into pGPTV-GUS-Kan. The construct obtained has the designation pGPTVI-myb11 :: GUS ( FIG. 7, construct III).

For a second shortened promoter, pBSK-myb11 is digested with EcoRV and NcoI. Overhanging ends are digested with T4 DNA polymerase and the plasmid is blunt-ended religated (as described in Sambrook et al., Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, 1989) to pBM-myb11 ( Fig to obtain. 9, construct X). The shortened insert is obtained from the vector pBM-myb11 by digestion with Hin dlii and SmaI, isolated on a 1% agarose gel and cloned into pGPTV-GUS-Kan. The construct obtained has the designation pGPTV2-myb11 :: GUS ( FIG. 7, construct IV).

For a third shortened promoter, pCR4-TOPO-myb11 is used as a template for a PCR. The HindIII and SmaI cleavage sites are introduced via the following primers

Forward primer 5'-AAGCTTAACCAATCAGGATTAAG-3 '(SEQ ID NO: 13) and

Reverse primer 5'-CCCGGGAAAATCACTCACTTCACT-3 '(SEQ ID NO: 12).

The fragment is isolated from a 1.5% agarose gel and cloned again into pGPTV-GUS-Kan. The construct obtained has the designation pGPTV3-myb11 :: GUS ( FIG. 8, construct V).

For a fourth shortened promoter, pCR4-TOPO-myb11 is used as a template for a PCR. The HindIII and SalI cleavage sites are introduced via the following primers

Forward primer 5'- AAGCTTGCATTATGGATTTTGTA-3 '(SEQ ID NO: 14) and

Reverse primer 5'-GTCGACAAAATCACTCACTTCACT-3 '(SEQ ID NO: 4).

The fragment is isolated from a 1.5% agarose gel and cloned again into pGPTV-GUS-Kan. The construct obtained has the designation pGPTV4-myb11 :: GUS-Kan ( FIG. 8, construct VI).

For a fifth shortened promoter, pCR4-TOPO-myb11 is used as a template for a PCR. The HindIII and XbaI cleavage sites are introduced via the following primers

Forward primer 5'-AAGCTTTGGACAATCATGTCATA-3 '(SEQ ID NO: 5) and

Reverse primer 5'-TCTAGAAAAATCACTCACTTCACT-3 '(SEQ ID NO: 6).

The fragment is isolated from a 1.5% agarose gel and cloned again in pGPTV-GUS-Kan. The construct obtained has the designation pGPTVS-myb11 :: GUS-Kan ( FIG. 8, construct VII).

For a sixth shortened promoter, pCR4-TOPO-myb11 is used as a template for a PCR. The HindIII and XbaI cleavage sites are introduced via the following primers.

Forward primer 5'-AAGCTTCTAATTAGAGACACTAGG-3 '(SEQ ID NO: 7) and

Reverse primer 5'-TCTAGAAAAATCACTCACTTCACT-3 '(SEQ ID NO: 6).

The fragment is isolated from a 1.5% agarose gel and cloned again in pGPTV-GUS-Kan. The construct obtained has the designation pGPTV6-myb11 :: GUS-Kan ( FIG. 8, construct VIII).

Example 6 Transformation and regeneration of transgenic Arabidop sis thaliana (Columbia) plants

Agrobacte is used to produce transgenic Arabidopsis plants rium tumefaciens (strain C58C1 pGV2260) with various myb11 Promoter-GUS vector constructs transformed. The agrobacteria strains are then used to produce transgenic plants used. For this purpose, a single transformed agrobacterial Colony in a 4 ml culture (medium: YEB medium with 50 μg / ml Kanamycin and 25 ug / ml rifampicin) incubated at 28 ° C overnight. This culture is then used to create a 400 ml culture in the inoculated into the same medium, incubated overnight (28 ° C, 220 rpm) and centrifuged (GSA rotor, 8,000 rpm, 20 min). The pellet is in infiltration medium (1/2 MS medium; 0.5 g / l MES, pH 5.8; 50 g / l sucrose) is resuspended. The suspension is in a Put in the plant box (Duchefa) and add 100 ml SILVET L-77 (with Polyalkylene oxide modified heptamethyltrisiloxane; Osi special ties Inc., Cat. P030196) was added to a final concentration of 0.02% added. The plant box with 8 to 12 plants is in a desiccator for 10 to 15 minutes and then a vacuum sender exposed to spontaneous ventilation. This will like 2 to 3 times repeated. Afterwards, all plants are placed in pots with moisture planted in soil and grown under long-day conditions (Tagestem temperature 22 to 24 ° C, night temperature 19 ° C; 65% relative air humidity). The seeds are harvested after 6 weeks.

Alternatively, transgenic Arabidopsis plants can be rooted transformation are obtained. White root shoots of a maximum 8 week old plants are used. For this purpose plants are under sterile conditions in 1 MS medium (1% sucrose; 100 mg / l inositol; 1.0 mg / L thiamine; 0.5 mg / L pyridoxine; 0.5 mg / l Nicotinic acid; 0.5 g MES, pH 5.7; 0.8% agar), used. Roots are grown on callus-inducing medium for 3 Days cultured (1 × Gambourg's B5 medium; 2% glucose; 0.5 g / l Mercaptoethanol; 0.8% agar; 0.5 mg / l 2,4-D (2,4-dichlorophenoxyes acetic acid); 0.05 mg / L kinetin). 0.5 cm long root sections who transferred to 10 to 20 ml of callus-inducing liquid medium (Composition as described above, but without the addition of agar) and with 1 ml of the overnight agrobacterial culture described above (grown at 28 ° C, 200 rpm in LB) inoculated and ge for 2 min shakes. The root explants become after removing over liquid medium by draining into callus-inducing medium transferred with agar, then in callus-inducing fluid sigmedium without agar (with 500 mg / l Betabactyl, SmithKline Beecham Pharma GmbH, Munich) and then incubated with shaking in shoot-inducing medium (5 mg / l 2-isopentenyl-adenine-phos phat; 0.15 mg / l indole-3-acetic acid; 50 mg / l kanamycin; 500 mg / l Betabactyl) transferred. After 5 weeks and 1 to 2 times the medium the small, green sprouts are switched to the germination medium (1 MS medium; 1% sucrose; 100 mg / l inositol; 1.0 mg / l thiamine; 0.5 mg / L pyridoxine; 0.5 mg / l nicotinic acid; 0.5 g MES, pH 5.7; 0.8% agar) and regenerated into plants.

Example 7 Detection of tissue-specific expression

To determine the properties of the promoter and who eat essential elements of the same, which make up its tissue specificity, to identify it is necessary to identify the promoter itself and different fragments of the same in front of a so-called reporter gene to set that allows a determination of the expression activity light. Bacterial β-glucuronidase may be mentioned as an example (Jefferson et al., EMBO J. 1987, 6, 3901-3907). The β-glucuroni This activity can be carried out in-planta by means of a chromogenic substrate tes like 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid in the frame activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405). For the investigations the tissue specificity, the plant tissue is cut, embedded, colored and analyzed as described (e.g. trees lein H et al., 1991 Mol Gen Genet 225: 121-128).

For the quantitative determination of activity is used as a substrate for uses the β-glucuronidase MUG (methylumbelliferyl glucuronide), which is split into MU (methylumbelliferone) and glucuronic acid. Under alkaline conditions, this cleavage can be quantitative can be followed fluorometrically (excitation at 365 nm, measurement of the Emission at 455 nm; SpectroFluorimeter BMG Polarstar +) like be written in Bustos M.M. et al., 1989 Plant Cell 1: 839-853.

SEQUENCE LOG

Claims (12)

1. Expression cassette for the transgenic expression of nucleic acids containing

• a) the promoter of the myb11 gene from Arabidopsis thaliana according to SEQ ID NO: 1, or
• b) functional equivalents or equivalent fragments of
• c) which have essentially the same promoter activities as a),

wherein a) or b) are functionally linked to a nucleic acid sequence to be expressed transgenically. 2. Expression cassette according to claim 1, characterized in that

• a) the nucleic acid sequence to be expressed is functionally linked to further genetic control sequences, or
• b) the expression cassette contains additional functional elements, or
• c) a) and b) are given.

3. Expression cassette according to one of claims 1 or 2, characterized in that the nucleic acid to be expressed sequence

• a) the expression of a protein encoded by said nucleic acid sequence, or
• b) enables the expression of a sense or anti-sense RNA encoded by said nucleic acid sequence.

4. Expression cassette according to claims 1 to 3, wherein the transgenically to be expressed nucleic acid sequence is selected from nucleic acids coding for amino acid transporters, Sac charid transporters, chalcone synthases, photolyases, photoreceptors gates, phytases, lipases, triacylglycerol lipases, acetyl-CoA Carboxylases, endochitinases, N-hydroxylating, multi functional cytochrome P-450, the transcriptional assets tor CBF1, invertases, the 2S albumin from Bertholletia ex celsa, endoxylglucan transferases, phytoene desaturases, the ABI3 protein, the VP-1 protein, "antifreeze" proteins, Glu tamate dehydrogenases, a late embryogenic gene (LEA), Cal cium-dependent protein kinases, calcineurins, farnesyltrans ferasen, ferritin, oxalate oxidase, DREB1A factor, trehalose phosphate synthase, trehalose phosphate phosphatase, trehalase, Cellulases, chitinases, glucanases, ribosome-inactivating Proteins, lysozymes, Bacillus thuringiensis endotoxin, a-Amy laser inhibitor, protease inhibitors, lectins, RMAases, ribo enzymes, fatty acid desaturases, fatty acid elongases, caffeinic acid O-methyltransferase, cinnamoyl alcohol dehydrogenase, ANT gene. 5. Expression cassette according to claims 1 to 4, wherein the transgenically to be expressed nucleic acid sequence is selected from the group of nucleic acid sequences with Genbank Ac cession numbers X92657, M20308, AJ002399, BAB00748, A19451, U62549, U40256, AB039325, L25042, S78423, U32624, U77378, V01311, AB044391, AF163819, X78815, AF306348, M60214, X68141, AJ222980, AF078796. 6. Vectors containing an expression cassette according to the An Proverbs 1 to 5. 7. Transgenic organism transformed with an expression cask Sette according to claims 1 to 5 or a vector according to An Proverb 6. 8. Transgenic organism according to claim 7 selected from the Group consisting of bacteria, yeast, fungus, animal and plant organisms. 9. Cell cultures, parts or transgenic reproductive material derived of a transgenic organism according to claims 7 or 8. 10. Use of a transgenic organism according to one of the claims che 7 to 8 or cell cultures derived therefrom, parts or transgenic propagation material according to claim 9 for the production processing of foodstuffs, animal feed, seeds, pharmaceuticals or fine chemicals. 11. Process for the production of pharmaceuticals or fine chemicals lien in transgenic organisms according to one of claims 7 or 8 or cell cultures derived therefrom, parts or transgenic propagation material according to claim 9, characterized records that the transgenic organism was bred and the ge desired pharmacon or the desired fine chemical iso is lated. 12. The method according to claim 11, wherein the fine chemicals enzymes, Vitamins, amino acids, sugars, saturated or unsaturated Fatty acids, natural or synthetic flavor, aroma or are dyes.