WO2002034924A2 - Expression cassette used for the transgenic expression of nucleic acids in the embryonic epidermis and/or flower of plants - Google Patents

Abstract

The invention relates to an expression cassette used for the transgenic expression of nucleic acids in the embryonic epidermis and/or the flower. Said expression cassette contains the promoter of the myb11 gene of Arabidopsis thaliana or functional equivalents or equivalent fragments thereof that have substantially the same promoter activity, said promoter being operably linked with a nucleic acid sequence that is to be transgenically expressed. The invention further relates to vectors derived from said expression cassettes. The invention also relates to transgenic plants transformed with said expression cassettes or vectors, to cultures, parts or transgenic propagation material derived therefrom and to the use thereof for producing foodstuff, feedstuff, seeds, pharmaceuticals or fine chemicals.

Description

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to control the expression of a particular gene in a transgenic plant and thus to achieve certain essential features of the plant. Various plant promoters are known.

For example, constitutive promoters such as the promoter 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 (1985), 810-812), the OCS ( Octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al., Plant Mol Biol 1995, 29: 637-646), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991 ). A disadvantage of these promoters is that they are constitutively active in almost all tissues of the plant. A targeted expression of genes in certain parts of plants or at certain times of development is not possible with these promoters.

Promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds are described. The stringency of the specificity as well as the expression activity of these promoters is very different. Promoters that ensure leaf-specific expression are to be mentioned, such as the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of the Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST- Potato LSI promoter (Stockhaus et al., EMBO J. 8 (1989), 2445-245).

Further promoters are, for example, promoters with specificity for tubers, storage roots or roots, such as, for example, the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato, the promoter of the starch synthase (GBSS1) or the sporamine promoter, fruit-specific promoters, such as the fruit-specific promoter made of tomato (EP-A 409625), fruit-ripening-specific promoters such as the fruit-specific promoter made of tomato

(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 stage of development of the plant is described, inter alia, in Baerson et al. (Baerson SR, Lamppa GK. Plant Mol Biol. 1993; 22 (2): 255-67).

Promoters are also known which control expression in seeds and plant embryos. For example, the promoters of genes that are used for storage proteins have been identified. encode different dicotyledons. Seed-specific promoters are, for example, the promoter of phaseoline (US 5,504,200, Bustos MM et al., Plant Cell. 1989; 1 (9): 839-53), of the 2S albuming gene (Joseffson LG et al., J Biol Chem 1987, 262 : 12196-12201), leguminum (Shirsat A et al., Mol Gen Genet. 1989;

215 (2): 326-331), of the USP (unknown seed protein; Bäumlein H et al., Molecular & General Genetics 1991, 225 (3): 459-67) of the Napin gene (Stalberg K, et al., L Planta 1996, 199: 515-519), the sucrose binding protein (WO 00/26388) or the LeB4 promoter (Baumlein H et al., Mol Gen Genet 1991, 225: 121-128). These control seed-specific expression of storage proteins.

However, the level of transgenic expression of heterologous genes under the control of these promoters is often highly dependent on the type of host plant. It was also found that the

Expression is rarely cell type specific. Differences in the expression pattern and the expression strength of a particular promoter can be caused by different host plants or by 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 within the seed has only been described for the seed coat: A cryptic promoter with specificity for the seed coat was identified by "T-DNA tagging" in tobacco (Fobert PR et al., Plant Journal 19946 (4): 567-77; US 5,824,863; WO 99/53067). Cryptic promoters or pseudopromotors are inherently inactive sequences within the genome, which, however, acquire expression-regulating functionality when positioned near genes.

The plant epidermis represents both the primary barrier and the essential contact surface of the plant organism with its environment. It has an important function in protecting the plant against biotic and abiotic stress factors, but also in the absorption of nutrients.

The epidermis-specific expression of certain genes is known. A lectin-like protein that accumulates in the shoot tip epider is the garden pea (Pisum safcivum) has been described (Maiti et al., Planta 1993, 190, 241-246). Sterk et al. describe a lipid transfer protein that is strongly expressed in the epidermal cells of carrot shoot tips (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 protein that is expressed in the epidermis of Pachyphy turn. Other examples of epidermis-specifically expressed genes, such as the Chalcon synthase and phenylalanine ammonia gene families have been reported (Schmelzer et al., Proc. Natl. Acad. Sci. USA 1988, 85, 2989-2993; Schmelzer et al., Plant Cell 1989, 1, 993-1001). Goodrich et al. describes enzymes of anthocyanin biosynthesis in the snapdragon flower, which are expressed in specialized, epidermal cells for a limited period during the development of the flower bud (Goodrich et al., Cell 1992, 68, 955-964). Wyatt et al. describes the proline-rich cell wall proteins SbPRPl, SbPRP2, and SbPRP2 from soy, which are expressed at certain stages of development in the epidermis, but at other times also in the vascular tissue (Wyatt et al., Plant Cell 1992, 4, 99-110).

Promoters are described with tissue specificity for the mesophyll and the pallisade cells in leaves (Broglie et al., Science 1984, 234, 838-845), the dividing shoot and the root meristem (Atanassova et al., Plant J. 1992 , 2, 291-300), pollen (Guerrero et al., Mol. Gen. Genet. 1990, 224, 161-168), seed endosperm (Stalberg et al., Plant Mol. Biol. 1993, 23, 20 671-683 ), Root epidermis (Suzuki et al., Plant Mol. Biol 1993, 21, 109-119), as well as for the root meristem, root vascular tissue and root nodules (Bogusz et al., Plant Cell 1990, 2, 633-641).

Epidermis-specific promoters are also known with activity in flowers (Koes et al., Plant Cell 1990, 2, 379-392) or after wound injuries (Liang et al., Proc. Natl. Acad. Sei. USA 1989, 86 , 9284-9288). No. 5,744,334 describes a plant promoter (Blec promoter) with specificity for the epidermis of the shoot tip. On the other hand, no promoter has been described that specifically ensures expression in the plant embryonic epidermis.

The expression of genes is regulated in all organisms via transcription factors. These also show development and

35 location-dependent expression patterns. Of particular interest is the family of R2R3-MYB transcription factors, which have conserved DNA binding domains comparable to that of the transcription factor c-myb and have been identified in almost all eukaryotes. It is believed that Arabidopsis thaliana is over 90

40 R2R3-MYB genes (Meissner RC et al., Plant Cell 1999; 11 (10): 1827-1840).

Despite the large number of known plant promoters, no promoter with specificity for the epidermis of the 45 plant embryo has been identified. Also are not promoters known that cause expression in the flower and in the epidermis of the plant embryo.

The task was therefore to identify corresponding promoters. This task was solved by providing the promoter for the Arabidopsis thaliana mybll gene. The promoter of the Arabidopsis thaliana mybll gene has an expression-regulating specificity which shows expression in the flower bud, the flower, the withering flower, the green pods and in the embryonic epidermis on day 4 of germination (compare FIGS. 1 to 6).

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

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 is a sensitive organ, especially against stress factors such as cold, and

b) the plant embryo is particularly sensitive and susceptible to biotic and abiotic stress factors, especially in the first days of embryogenesis.

The epidermis of the plant embryo has a special function because it is the immediate barrier and contact point with the environment.

A first subject of the invention therefore relates to an expression cassette for the transgenic expression of nucleic acids in the embryonic epidermis and / or containing 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) which have essentially the same promoter activities as a), where a) or b) are functionally linked to a nucleic acid sequence to be expressed transgenically.

Expression cassette for the transgenic expression of nucleic acids means all such constructions which have been obtained by genetic engineering methods and in which either

(a) the promoter specific for the embryonic epidermis and / or the flower or a functional equivalent or fragment thereof derived therefrom, or

b) the nucleic acid sequence to be expressed, or

c) (a) and (b)

are not in their natural, genetic environment (i.e. at their natural chromosomal locus) or have been modified by genetic engineering methods, the modification being, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.

In the context of the expression cassette according to the invention, the expression of a specific nucleic acid by a promoter with specificity for the embryonic epidermis and / or the flower can lead to the formation of sense RNA or anti-sense RNA. As a result, the sense RNA can be translated into certain polypeptides. The expression of certain genes can be downregulated with the anti-sense RNA.

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, the nucleic acid sequence to be expressed transgenically and if necessary. further regulatory elements such as a terminator such that each of the regulatory elements can fulfill its function in the transgenic expression of the nucleic acid sequence, depending on the arrangement of the nucleic acid sequences to sense or anti-sense RNA. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically is positioned behind the sequence functioning as a promoter, so that both sequences are covalently linked to one another. The distance between the promoter sequence and the transgene is preferably to be expressed. Nucleic acid sequence less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

The creation of a functional link can be implemented using common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. 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). However, further sequences can also be positioned between the two sequences, which for example have the function of a linker with certain restriction enzyme interfaces or a signal peptide. The insertion of sequences can also lead to the expression of fusion proteins.

Various ways are known to the person skilled in the art in order to arrive at an expression cassette according to the invention. An expression cassette according to the invention is preferably produced, for example, by direct fusion of a nucleic acid sequence acting as a promoter with specificity for the embryonic epidermis and / or the flower with a nucleotide sequence to be expressed. Common recombination and cloning techniques, such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. 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). The expression cassette, consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector according to the invention and inserted into a plant genome by, for example, transformation.

JAn expression cassette is also to be understood as such constructions in which the promoter, without having been functionally linked to a nucleic acid sequence to be expressed, is introduced, for example via targeted homologous recombination or a random insertion into a host genome, there regulatory control over then takes over functionally linked nucleic acid sequences and controls their transgenic expression. By inserting the promoter - to

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al., Plant Mol Biol Rep 1992 10: 324-414) or the ß-galactosidase, the ß-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907) is very particularly preferred.

Otherwise unchanged conditions means that the expression which is initiated by one of the expression cassettes to be compared is not modified by combination with additional genetic control sequences, for example enhancer sequences.

Vectors according to the invention also contain the expression cassettes described above.

The nucleic acid sequences contained in the expression cassettes or vectors according to the invention can be functionally linked to further genetic control sequences in addition to the promoter with specificity for the embryonic epidermis and / or the flower. The term “genetic control sequences” is to be understood broadly and means all those sequences which have an influence on the formation or the function of the expression cassette according to the invention. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms. The expression cassettes according to the invention preferably comprise 5 'upstream of the respective nucleic acid sequence to be expressed transgenically, the promoter with specificity for the embryonic epidermis and / or the flower and 3' downstream a terminator sequence as an additional genetic control sequence, and, if appropriate, further customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be expressed transgenically.

Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. For example, through genetic control sequences, tissue-specific expression can also depend on certain stress factors. Corresponding elements are, for example, for water stress, abscisic 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).

Furthermore, other promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria. In principle, all promoters described above can be used as plant promoters. For example, it is conceivable that a certain nucleic acid is transcribed by a promoter (for example the promoter with specificity for the embryonic epidermis and / or the flower) in a plant tissue as sense RNA and translated into the corresponding protein, while the same nucleic acid sequence is translated by another promoter with another Specificity in another tissue is transcribed to anti-sense RNA and the corresponding protein is downregulated. This can be achieved by means of an expression cassette according to the invention, in that one promoter is positioned in front of the nucleic acid sequence to be expressed transgenically and the other promoter behind it.

Genetic control sequences also include the 5'-untranslated region, introns or the non-coding 3 'region of genes, preferably the mybll gene from Arabidopsis thaliana. It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. They can also promote tissue specificity (Rouster J et al., Plant J. 1998, 15: 435-440.). Conversely, the 5 'untranslated region of the opaque-2 gene suppresses expression. 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 sequence given under SEQ ID N0: 1 contains the section of the mybll gene which represents the promoter and the 5 'untranslated region up to the ATG start codon of the MYBll transcription factor.

The expression cassette can advantageously one or more so-called "enhancer sequences" functionally linked to the

Contain promoters that allow increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically. The nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct.

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or function of the expression cassettes, vectors or transgenic organisms according to the invention. Examples include, but are not limited to:

a) Selection markers which are resistant to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. to lend.

b) reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or of the expression site or time. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (l): 29-44) such as "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), the β-galactosidase, the β-glucuronidase is particularly preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907).

c) Replication origins, which are an increase in the expression cassettes or vectors according to the invention in, for example

Ensure E.coli. Examples include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sa-brook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) ,

d) Elements which are required for an agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.

For the selection of successfully homologously recombined or also transformed cells, it is generally necessary to additionally introduce a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglu cose-6-phosphate (WO 98/45456) or an antibiotic. The selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al., Plant Cell Reports 5 (1986), 81-84).

Arabidopsis thaliana. Functionally equivalent sequences also include all the sequences which are derived from the complementary counter strand of the sequence defined by SEQ ID NO: 1 and which have essentially the same promoter activity.

Functional equivalents initially mean DNA sequences which hybridize under standard conditions with the nucleic acid sequence described by SEQ ID NO: 1 or the nucleic acid sequence complementary to it and which have essentially the same promoter activities as the nucleic acid sequence described by SEQ ID N0: 1. Standard hybridization conditions are to be understood broadly and mean stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al. , in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 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 approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C) (20X SSC: 0.3M sodium citrate, 3MNaCl, pH 7.0). Furthermore, the temperature during the washing step can be raised 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 simultaneously, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as for example amide or SDS can also be used during the 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

(i) 4X SSC at 65 ° C, or

(ii) 6X SSC at 45 ° C, or

(iii) 6X SSC at 68 ° C, 100μg / ml denatured fish sperm DNA, or (iv) 6X SSC, 0.5% SDS, 100μg / ml denatured, fragmented salmon sperm DNA at 68 ° C, or

(v) 6X SSC, 0.5% SDS, 100μg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42 ° C.

(vi) 50% formamide, 4XSSC at 42 ° C, or

(vii) 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 rate at 42 ° C, or

(viii) 2X or 4X SSC at 50 ° C (weakly stringent condition), or

(ix) 30 to 40% formamide, 2X or 4X SSC at 42 ° C (weakly stringent condition).

(2) washing steps with for example

(i) 0.015 MNaCl / 0.0015 M sodium citrate / 0.1% SDS at 50 ° C; or

(ii) 0.1XSSC at 65 ° C, or

(iii) 0.1X SSC, 0.5% SDS at 68 ° C, or

(iv) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C, or

(v) 0.2X SSC, 0.1% SDS at 42 ° C, or

(vi) 2X SSC at 65 ° C (weakly stringent condition).

According to the invention, preference is given to using such nucleic acids in the expression cassettes or from vectors derived therefrom which have the double-stranded DNA sequence described by SEQ ID NO: 1 or fragments thereof, such as, for example, the double-stranded DNA sequences described by SEQ ID NO: 2 and 15 to 19 DNA sequences hybridize under the conditions defined above and have essentially the same promoter activity as.

Sequence fragments derived from SEQ ID NO: 1 are particularly preferred, which comprise base pairs 1700 to 2700 of the sequence with SEQ ID NO: 1. A functional equivalent is also understood to mean, in particular, natural or artificial mutations of the mybll promoter described under SEQ ID NO: 1 and its homologs from other plant genera and plant species, which continue to initiate expression in the plant embryonic epidermis and / or flowers can. Mutations include substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. Thus, for example, the present invention also encompasses those nucleotide sequences which are obtained by modification of the mybll promoter nucleotide sequence in accordance with SEQ ID NO: 1. The aim of such a modification can be, for example, the further limitation of the sequence contained therein to certain regulatory elements or, for example, the insertion of further restriction enzyme interfaces, the removal of superfluous DNA or the addition of further sequences, for example further regulatory sequences.

The sequence can also be limited to specific, essential regulatory regions with the aid of a search routine for searching promoter elements. Certain promoter elements are often present in abundance in the regions relevant to promoter activity. This analysis can be carried out, for example, using computer programs such as the PLACE program ("Plant Cis-acting Regulatory DNA Elements") (K.Higo et al., (1999) Nuclear Acids Research 27: 1, 297-300).

Where insertions, deletions or substitutions, e.g. Transitions and transversions can be used, techniques known per se, such as in vitro mutagenesis, primer repair, restriction or ligation, can be used. Through manipulations, such as Restriction, chewing-back or filling of overhangs for blunt ends, complementary ends of the fragments can be made available for the ligation. Analogous results can also be obtained 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 set by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) the following parameter is calculated:

Gap Weight: 12 Length Weight: 4

Average Match: 2,912 Average Mismatch: -2, 003 A sequence which has a homology of at least 20% on a nucleic acid basis with the sequence SEQ ID NO. 1, understood a sequence which, when compared with the sequence SEQ ID NO. 1 according to the above program algorithm with the above parameter set has a homology of at least 20%.

Functional equivalents derived from one of the promoter sequences used in the expression cassettes or vectors according to the invention, for example by substitution, insertion or deletion of nucleotides, have a homology of at least 20%, preferably 40%, preferably at least 60%, particularly preferably at least 80%, very particularly preferably at least 90%, and are distinguished by essentially the same transcription activity compared to the mybll promoter sequence according to SEQ-ID NO: 1.

Further examples of the promoter sequences used in the expression cassettes or expression vectors according to the invention can be found, for example, from various organisms whose genomic sequence is known, for example from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthinum, by comparing homology easy to find from databases.

Functional equivalents also include those promoter variants whose function is weakened or enhanced compared to the starting promoter.

Further suitable functionally equivalent expression cassettes are sequences which express a fusion protein under the control of the promoter sequence, one component of the fusion protein the nucleic acid sequence to be expressed, the other a regulatory protein sequence, e.g. is a signal or transit peptide that directs the gene product to the desired site of action.

An expression cassette according to the invention is produced, for example, by fusion of the mybll promoter according to SEQ ID NO: 1 or a functional equivalent with a nucleotide sequence to be expressed, optionally a sequence coding for a transit peptide, preferably a chloroplast-specific transit peptide, which is preferably between the promoter and the respective nucleotide sequence is arranged, and a terminator or polyadenylation signal. Common recombination and cloning techniques are used for this, such as those used in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labo- ratory, Cold Spring Harbor, NY (1989) as well as in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, FM et al. , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

The specificity of the expression constructs and vectors according to the invention for the plant, embryonic epidermis and the flower is particularly advantageous. The plant epidermis represents both the primary barrier and the essential contact surface of the plant organism with its environment. It has an important function in protecting the plant against biotic and abiotic stress factors. Biotic stress factors can be pathogens, bite from animals etc., abiotic stress can

Heat, cold, dryness, UV light etc. The epidermis also has a function in attracting beneficial insects through pigment storage or the synthesis of volatile chemicals, it provides mechanical stability and prevents water loss. The epidermis also has an essential role in the absorption of water, gases and nutrients in the semen embryo.

The plant's natural defense mechanisms, for example against pathogens, are often inadequate. The introduction of foreign genes from plants, animals or microbial sources can strengthen the immune system. Examples are protection against insect damage in tobacco by expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37) or protection of the tobacco against fungal attack by expression of a chitinase the bean under the control of the CaMV promoter (Broglie et al., Science 1991, 254, 1194-1197).

Cold spells during flowering lead to significant crop losses every year. A targeted expression of protective proteins in the flowering period can provide protection.

For a high efficiency of such genetic engineering approaches, concentrated expression of the corresponding nucleic acid sequence to be expressed transgenically is particularly advantageous in the outermost envelope of the plant, the epidermis, or the flower. Constituent expression in the entire plant can question the effect, for example by diluting it, or impair the growth of the plant or the quality of the plant product. In addition, constitutive expression can lead to an increased shutdown of the transgene ("gene silencing"). For this purpose promoters with specificity for the embryonic epidermis and / or the blossom are advantageous.

A large number of proteins are known to the person skilled in the art, the recombination of which in the embryonic epidermis or the blossom is advantageous. Furthermore, a large 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 also advantageous effects can be achieved. Not exemplary, however

¹⁰ restrictive for advantageous effects are: The achievement of resistance to abiotic stress factors (heat, cold, dryness, increased humidity, environmental toxins, UV radiation) and biotic stress factors (pathogens, viruses, insects and diseases), the improvement of food or feed egg

I ⁵ properties, improving the growth rate or yield, achieving a longer or earlier flowering period, changing or enhancing the fragrance or coloring of the flowers. For the nucleic acid sequences or polypeptides that can be used in these applications, examples, but not

20 restrictive:

1. Improved UV protection of the plant embryo by changing the pigmentation through expression of certain polypeptides such as enzymes of flavonoid biosynthesis such as the chalcon synt

25 rabbit or the phenylalanine ammonia or certain enzymes of DNA repair such as photolyase (Sakamoto A et al., DNA Seq. 1998; 9 (5-6): 335-40). Nucleic acids which are particularly preferred are those for the chalcone synthase from Arabidopsis thaliana (GenBank Acc.-No .: M20308), the 6-4 photolyase from Arabidopsis

³⁰ sis thaliana (GenBank Acc. -No .: BAB00748) or the blue light photoreceptor / photolyase homolog (PHHl) from Arabidopsis thaliana (GenBank Acc.-No .: U62549) or functional equivalents thereof.

³⁵ 2. Improved protection of the plant embryo or the flower against abiotic stress factors such as drought, heat or cold, for example by overexpression of the "antifreeze polypeptides from Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thaliana Trans- ⁰ activation activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), a late embryogenetic gene (LEA), for example from barley (WO 97/13843), calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), farnesyl transferases (WO 99/06580), Pei ZM et al., Science 1998, 282:

rf^ ⁴⁵ 287-290), ferritin (Deak M et al., Nature Biotechnology 1999, 17: 192-196), oxalate oxidase (WO 99/04013; Dunwell JM Biotechnology and Genetic Engineering Reviews 1998, 15: 1-32),

rf ^

Secticidal proteins such as Bacillus thuringiensis endotoxin, amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), glucanases, lectins such as phytohemagglutinin, snowdrop lectin, wheat germ agglutinin, RNAsen or ribozymes. Nucleic acids which are particularly preferred for the chit42 endochitinase from Trichoderma harzianum (GenBank Acc.- No.: S78423) or for the N-hydroxylating, multifunctional cytochrome P-450 (CYP79) from Sorghum bicolor (GenBank Acc.-No. : U32624) or encode their functional equivalents.

6. Achieving an insect defense or attracting, for example, by increased release of volatile fragrance or messenger substances by, for example, enzymes of terpene biosynthesis.

7. Achieving storage capacity in the epidermis cells, which normally do not contain any storage proteins or lipids, with the aim of increasing the yield of these substances, for example by expression of an acetyl-CoA carboxylase. Preference is given to nucleic acids which code for Medicago sativa acetyl-CoA carboxylase (Accase) (GenBank Acc.-No .: L25042) or functional equivalents thereof.

8. Expression of transport proteins which improve the uptake of metabolites, nutrients or water into the embryo and thus optimize embryo growth, the metabolite composition or the yield, for example by expression of an amino acid transporter which accelerates the uptake of amino acids, or a monosaccharide -Transporter that promotes the absorption of sugars. Are preferred

Nucleic acids which code for the cationic amino acid transporter from Arabidopsis thaliana (GenBank Acc.-No .: X92657) or for the monosaccharide transporter from Arabidopsis thaliana (GenBank Acc.-No .: AJ002399) or functional equivalents of the same.

9. Expression of genes which cause the accumulation of fine chemicals, such as tocopherols, tocotrienols or carotenoids, in the epidermis of the seed. The phytoendesaturase may be mentioned as an example. Preference is given to nucleic acids which code for the phytoendesaturase from Narcissus pseudonarcissus (GenBank Acc. No .: X78815) or functional equivalents thereof.

10. Modification of wax ester formation or the composition of the stored oligosaccharides to improve protection against environmental influences or to improve digestion Availability when used in feed or food. The overexpression of endoxyloglucan transferase may be mentioned as an example. Preferred are nucleic acids which code for the endoxyloglucan transferase (EXGT-Al) from Arabidopsis thaliana (Gen-Bank Acc. -No.: AF163819) or functional equivalents thereof.

11. Prolongation or abolition of the resting phase by expression of transcription factors which intervene in the regulation of genes relevant to dormancy. Examples include the overexpression of the ABI3 protein (abscisic acid insensitive gene) or the Viviparous-1 protein (VP-1). The germ time change can result in an increase in yield and a shortened embryonic development. Preferred nucleic acids are those 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 functional equivalents encode them.

12. Generation of sterile plants by preventing fertilization 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 fatty acids such as arachidonic acid or EP (eicosapentaenoic acid) or DHA (docosahexaenoic acid) by expression of fatty acid elongases and / or desaturases or production of proteins with improved nutritional value such as a high proportion of essential amino acids ( eg the methionine-rich 2S albumingens of the Brazil nut). Preferred nucleic acids are those for the methionine-rich 2S 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 (Sakuradani et al 1999 Gene 238: 445-453), the Δ5-desaturase from Caenorhabditis 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-desaturase from Mortierella alpina (Michaelson et al. JBC 273: 19055 - 19059) , the Δ6 elongase from Caenorhabditis elegans (Beaudoin et al. 2000, PNAS 97: 6421-6426), the Δ6 elongase from Physcomitrella patens (Zank et al. 2000, Biochemical Society Transactions 28: 654-657) or functional Encode equivalents thereof. 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 Immuno1. 1999; 236: 275-92.

15. Modification of the fiber content in seed-based foods by, for example, expression of antisense transcripts of the caffeine acid-O-methyltransferase or the cinnamoyl alcohol-containing hydrogenase gene.

16. Inhibition of the maturation of the seed coat by expression of, for example, ribonuclease genes or transcription factors in order to achieve an increased seed size, to reduce the relative biomass of the seed coat and to facilitate the unveiling of the seed. An overexpression of the ANT (AINTEGUMENTA) gene may be mentioned here as an example. Preferred are nucleic acids which code for the AINTEGUMENTA gene from Arabidopsis thaliana (GenBank Acc.-No .: U40256) or functional equivalents thereof.

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

Functional equivalents here - analogous to the definition of the functional equivalents of the promoter with specificity for the embryonic epidermis and / or the flower - mean all the sequences which have essentially the same function, i.e. are capable of the function (for example a substrate conversion or a signal transduction) as well as the protein mentioned by way of example. The functional equivalent may well differ in other features. For example, it may have a higher or lower activity, or it may have other functionalities.

Functional equivalents also means sequences which code for fusion proteins consisting of one of the preferred proteins and other proteins, for example another preferred protein or else a signal peptide sequence.

The person skilled in the art is also aware that he does not have to express the genes described above directly using the nucleic acid sequences coding for these genes or to repress them, for example, by antisense. For example, he can also use artificial transcription factors of the zinc finger protein type (Beerli RR et al., Proc Natl Acad Sei USA 2000; 97 (4): 1495-500). These factors are reflected in the regulatory areas of endogenous genes to be expressed or repressed and, depending on the design of the factor, cause expression or repression of the endogenous gene.

Another object of the invention relates to transgenic organisms, transformed with at least one expression cassette according to the invention or a vector according to the invention, as well as cells, cell cultures, tissues, parts - such as leaves, roots etc. in plant organisms - or well reproduced from such organisms.

Organism, starting or host organisms are understood to mean prokaryotic or eukaryotic organisms, such as, for example, microorganisms or plant organisms. Preferred microorganisms are bacteria, yeast, algae or fungi.

Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or cyanobacteria, for example of the genus Synechocystis. Especially preferred are microorganisms which are capable of infecting plants and thus of transmitting the constructs according to the invention. Preferred microorganisms are those from the genus Agrobacterium and in particular from the species Agrobacterium tumefaciens.

Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.

Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6 described mushrooms.

Plants are primarily host or parent organisms preferred as transgenic organisms. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Also included are the mature plants, seeds, sprouts and seedlings, as well as parts, propagation material and cultures derived therefrom, for example cell cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.

Annual, perennial, monoeotyledonous and dicotyledonous plants are preferred host organisms for the production of transgenic plants. The expression of genes in the embryonic epidermis and especially the flower is also advantageous for all jewelry plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawn. Angiosperms, bryophytes such as Hepaticae may be mentioned as examples but not as restrictive

(Liverwort) and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, - algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae

(Dietomeen) and Euglenophyceae.

Plants within the scope of the invention include, by way of example and not limitation, the families of the Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as carnations, Solanaceae such as petunias, Gesneriaceae such as the African violet and Balsamineaaceae such as Balsamineaaceae and Balsamineaaceae , Iridaceae like gladiolus, iris, freesia and crocus, Compositae like marigold, Geraniaceae like geraniums, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others.

The families of the leguminosae such as pea, alfalfa and soy are to be mentioned as examples but not restrictive for flowering plants; Gramineae such as rice, corn, wheat; Solanaceae such as tobacco and others; the Umbelliferae family, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (selaria)) and others; the family of Solanacea, especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) and others; and the genus Capsicum, especially the species annum (pepper) and others, - the family of the Leguminosae, especially the genus Glycine, especially the species max (soybean) and others; and the Cruciferae family, especially the Brassica genus, especially the campestris (turnip), 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 Compositae family, especially the Lactuca genus, especially the Sativa species (lettuce) and others.

The transgenic plants according to the invention are selected in particular from monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, millet, rye, triticale, maize, rice or oats, and sugar cane. Furthermore, the transgenic plants according to the invention are selected in particular from dicotyledonous crop plants, such as, for example Brassicacae such as rapeseed, cress, arabidopsis, cabbage or canola, leguminosae such as soy, alfalfa, pea, alfalfa, bean family or peanut

Solanaceae such as potato, tobacco, tomato, eggplant or paprika, Asteraceae such as sunflower, tagetes, lettuce or calendula, Cucurbitaceae such as melon, pumpkin or zucchini, as well as linen, cotton, hemp, flax, red pepper, carrot, carrot, sugar beet and the various Tree, nut and wine species.

Arabidopsis thaliana, Nicotiana tabacum, Tagetes erecta, Calendula officinalis and all genera and species that are used as food or feed, such as the cereals described, or are suitable for the production of oils, such as oilseed rape (for example rape), are particularly preferred ), Types of nuts, soy, sunflower, pumpkin and peanut.

Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae or cyanobacteria, and mosses.

Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.

The production of a transformed organism or cell requires that the corresponding DNA be introduced into the corresponding host cell. A large number of methods are available for this process, which is referred to as transformation (see also Keown et al. 1990 Methods in Enzymology 185: 527-537). For example, the DNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. The cell can also be chemically permeabilized, for example with polyethylene glycol, 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. Electroporation is another suitable method for introducing DNA, in which the cells are reversibly permeabilized by an electrical impulse.

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

In addition to these "direct" transformation techniques, a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant after agrobaterium infection. Part of this plasmid, called T-DNA (transferred DNA), is integrated into the genome of the plant cell.

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

The introduction of an expression cassette according to the invention into cells, preferably into plant cells, can advantageously be implemented using vectors. Vectors can be, for example, plasmids, cosmids, phages, viruses or even agrobacteria.

In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. Preferred vectors are those which enable stable integration of the expression cassette into the host genome.

In the case of injection or electroporation of DNA into plant cells, there are no special requirements for the plasmid used. Simple plasmids such as the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary that there is an additional selectable marker gene on the plasmid.

Transformation techniques are described for various monocot and dicotyledonous plant organisms. Various possible plasmid vectors are also available for the introduction of foreign genes into plants, which generally contain an origin of replication for reproduction in E. coli and a marker gene for 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 site. The resulting plasmid is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant plasmid obtained using methods familiar to the person skilled in the art. Restriction analysis and sequencing can be used to check the cloning step.

Transformed cells i.e. Those that contain the inserted DNA integrated into the DNA of the host cell can be selected from untransformed ones if a selectable marker is part of the inserted DNA. Any gene that can confer resistance to antibiotics or herbicides can act as a marker, for example. Transformed cells,

Those expressing such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type. Examples are the bar gene that confers resistance to the herbicide phosphinotricin (Rathore KS et al., Plant Mol Biol.

15 1993 Mar; 21 (5): 871-884), the nptll gene, which confers resistance to kanamycin, the hpt gene, which confers resistance to hygro ycin, or the EPSP gene, which confers resistance to the herbicide glyphosate.

20 Depending on the method of introducing DNA, additional genes may be required on the vector plasmid. If Agrobacteria is used, the expression cassette must be integrated in special plasmids, either in an intermediate vector (English: shuttle or intermediate vector) or in a binary vector. If for example

If a Ti or Ri plasmid is to be used for transformation, at least the right boundary, but mostly the right and left boundary of the Ti or Ri plasmid T-DNA as the flanking region, is connected to the expression cassette to be inserted. Binary vectors are preferably used. Binary vector

30 ren can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA restriction sequences. They can be transformed directly into Agrobacterium (Holsters et al., Mol. Gen. Genet. 163 (1978),

35 181-187). The selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin. The Agrobacterium, which acts as the host organism in this case, should already contain a plasmid with the vir region. This is for the transfer

40 g of T-DNA required on the plant cell. An Agrobacterium transformed in this way can be used to transform plant cells.

The use of T-DNA for the transformation of plant cells 45 has been intensively investigated and described (EP 120516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Be., 4: 1-46 and An et al. , EMBO J. 4 (1985) 277-287). Various binary vectors are known and some are commercially available, for example pBIN19 (Clontech Laboratories, Inc. USA).

To transfer the DNA to the plant cell, plant explants are co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Starting from infected plant material (e.g. leaf, root or stem parts, but also protoplasts or suspensions of plant cells), whole plants can be regenerated using a suitable medium, which can contain, for example, antibiotics or biocides for the selection of transformed cells. The plants obtained can then be screened for the presence of the introduced DNA, here the expression cassette according to the invention. As soon as the DNA is integrated into the host genome, the corresponding genotype is generally stable and the corresponding insertion is also found in the subsequent generations. As a rule, the integrated expression cassette contains a selection marker which gives the transformed plant resistance to a biocide (for example a herbicide) or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. The selection marker allows the selection of transformed cells from untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84). The plants obtained can be grown and crossed in a conventional manner. Two or more generations should be cultivated to ensure that genomic integration is stable and inheritable.

The methods mentioned are described, 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 (1991), 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711).

Once a transformed plant cell has been made, a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown. The effectiveness of the expression of the transgenically expressed nucleic acids can be determined, for example, in vitro by sprout meristem propagation using one of the selection methods described above.

According to the invention are also derived from the transgenic organisms described above, cell cultures, parts - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits.

Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as food or feed, for example directly or after preparation known per se.

Another object of the invention relates to the use of the transgenic organisms according to the invention described above and the cells, cell cultures, parts derived therefrom - such as roots, leaves etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits for the production of food or feed, pharmaceuticals or fine chemicals.

Also preferred is a process for the recombinant production of pharmaceuticals or fine chemicals in host organisms, wherein a host organism is transformed with one of the expression cassettes described above and this expression cassette contains one or more structural genes which code for the desired fine chemical or the biosynthesis of the desired fine chemical catalyze, the transformed host organism is grown and the desired fine chemical is isolated from the growth medium. This process is widely applicable to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavors, aromas and colors. The production of tocopherols and tocotrienols and carotenoids is particularly preferred. The transformed host organisms and the isolation from the host organisms or from the growth medium are cultivated using methods known to those skilled in the art. The production of pharmaceuticals, such as antibodies or vaccines, is described by Hood EE, Jilka JM. Curr Opin Biotechnol. 1999 Aug; 10 (4): 382-6; Ma JK, Vine ND.Curr Top Microbiol Immuno1. 1999; 236: 275-92. sequences

1. SEQ ID NO: 1 promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene.

2. SEQ ID NO: 2 2035bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll 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 primers, reverse primer Z17R3

10. SEQ ID NO: 10 oligonucleotide primers, PCR adapter primer 1

11. SEQ ID NO: 11 oligonucleotide primer, myBLI forward primer

12. SEQ ID NO: 12 oligonucleotide primers, myBLI reverse primer

13. SEQ ID NO: 13 oligonucleotide primers

14. SEQ ID NO: 14 oligonucleotide primers

15. SEQ ID NO: 15 888bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene.

16. SEQ ID NO: 16 282bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene

17. SEQ ID NO: 17 1394bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene

18. SEQ ID NO: 18 1191bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene 19. SEQ ID NO: 19 665bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene

pictures

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

1. Fig. 1 (I and II) show a section through a seed and thus through the plant embryo (Arabidopsis thaliana). "in" denotes the integument, "h" the hypocotyl, "c" the cotyledon and "r" the root. Fig.l (I) shows the section without clarifying the expression region. Fig.l (II) illustrates the expression region of the mybll 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 scar papillae (English: 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 clarifying the expression region. Fig. 2 (II) illustrates the expression region of the mybll promoter, which is found strongly in the hatched regions, less strongly in the dotted regions. These regions correspond on the one hand to the ovary wall and on the other hand to the ovaries themselves.

3. Fig. 3 (I and II) show a section through the ovary systems of Arabidopsis thaliana. "al" denotes the pollen sac

(English: anther locules), "g" the gynoecium, "op" the egg system (English: ovule primordia) and "sp" a petal (sepals). Fig. 3 (I) shows the section without clarifying the expression region. Fig. 3 (II) illustrates the expression region of the mybll promoter, which can be found in the white hatched regions (marked by white arrows). These regions correspond to the egg facilities.

4. Fig. 4 (I and II) show a section through the plant flowers (Arabidopsis thaliana). "im" denotes the flower meristem (tunica). The numbers 2 and 6 describe flowers in the flower development stage 2 or 6 (Arabidopsis Atlas of Morphology, Bowman J ed ^' ., Springer Verlag, New York, USA, 1995). Fig. 4 (II) shows the section without clarifying the expression region. Fig. 4 (I) illustrates the express sion region of the mybll 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 plant and the petal primordia (development stage 6).

5. Fig. 5 (I) shows a schematic representation of the expression in the flower stage 2. "fa" denotes the flower tip (floral apex). The expression region of the mybll promoter can be found in the hatched region. 5 (II) shows a schematic representation of the expression in the flower stage 6. "p" denotes the petal primordium, "ms" denotes the stamen plant), "g" the gynoecium. The expression region of the mybll promoter can be found in the hatched region

6. FIG. 6: The result of an RT-PCR is shown using different tissues to determine the expression pattern of the mybll promoter. Expression can be demonstrated in the seedling (day 4), the flower bud, flowering and withering flower, and the green pods.

7. Fig. 7: Schematic representation of the vectors pCR-TOPO-mybll (I), pGPTV-ybll:: GUS (II), pGPTVl-mybll :: GUS (III) and pGPTV2-mybll:: GUS (IV). The direction of the arrow indicates the orientation of the promoter, with the arrow pointing in the direction of the start codon. The abbreviations have the following meaning: mybll: promoter or promoter fragment of the mybll promoter NOS-T: terminator sequence of nopaline synthase (NOS) GUS: reporter gene (bacterial β-glucuronidase)

8. Fig. 8: Schematic representation of the vectors pGPTV3-mybll :: GUS (V), pGPTV4-mybll:: GUS (VI), pGPTV5-mybll :: GUS

(VII) and pGPTV6-mybll:: CIS (VIII). The direction of the arrow indicates the orientation of the promoter, with the arrow pointing in the direction of the start codon. The abbreviations have the following meaning: mybll: promoter or promoter fragment of the mybll promoter NOS-T: terminator sequence of nopaline synthase (NOS) GUS: reporter gene (bacterial β-glucuronidase)

9. Fig. 9: Schematic representation of the vectors pBSK-mybll (IX) and pBM-ybll (X). The direction of the arrow indicates the orientation of the promoter, with the arrow pointing in the direction of the start codon. The abbreviations have the following meaning: mybll: promoter or promoter fragment of the mybll promoter Examples

General methods:

The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The cloning steps carried out in the context of the present invention, such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA are carried out as in Sambrook et al , (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules takes place with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).

Example 1: Plant growth conditions for tissue-specific RT-PCR analysis

In order to obtain seedlings 4 or 7 days old, approximately 400 seeds (Arabidopsis thaliana ecotype Columbia) are sterilized on the surface with an 80% ethanol solution for 2 minutes, treated with a sodium hypochlorite solution (0.5% v / v) for 5 minutes, Washed three times with distilled water and incubated at 4 ° C for 4 days to ensure even germination. The seeds are then placed on petri dishes with MS 'medium

(Sigma M5519) with the addition of 1% sucrose, 0.5 g / 1 MES (Sigma M8652), 0.8% Difco-BactoAgar (Difco 0140-01), pH 5.7. The seedlings are grown in a 16-hour light / 8-hour dark cycle (Philips 58W / 33 white light lamps) at 22 ° C and harvested 4 or 7 days after the start of the germination phase.

100 seeds for the extraction of ^Roots sterilized as described above, incubated for 4 days at 4 ° C and then see in 250ml bottles with MS medium (Sigma M5519) with the addition of a further 3% sucrose and 0.5 g / 1 MES (Sigma M8652), pH 5.7. The seedlings are grown in a 16-hour light / 8-hour dark cycle (Philips 58W / 33 white light lamps) at 22 ° C, 120 rpm and harvested after 3 weeks. For all other plant organs used, the seeds are sown on uniform soil (type VM, Manna-Italia, Via S. Giaco o 42, 39050 San Giacomo / Laives, Bolzano, Italy), incubated for 4 days at 4 ° C to ensure uniform germination and then grown in a 16-hour light / 8-hour dark cycle (OSRAM Lumi- lux Daylight 36W / 12 fluorescent tubes) at 22 ° C. Young rosette leaves are harvested in the 8-leaf stage (after 3 weeks), mature rosette leaves are harvested after 8 weeks just before

Harvested stems. Inflorescences (apices) of the imposing stems are harvested shortly after imposition. Stems, stem leaves and flower buds are harvested in development stage 12 J Bowmann J (ed.), Arabidopsis, Atlas of Morphology, Springer New York, 1995) before stamen development. Opened

Flowers are harvested in stage 14 immediately after stamen development. Wilting flowers are harvested in stages 15 to 16. The green and yellow pods used had a length of 10 to 13 mm.

Example 2: RJMA 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 DNasel (15 units, Boehringer, Mannheim) before the cDNA synthesis. The first strand cDNA synthesis is carried out starting from 6μg total RNA with an oligo (dT) primer and RT Superscript ™ II enzyme (300 Uήits) according to the manufacturer in a total volume of 20 μl (Life Technologies, Gaithersburg, MD). For this purpose, 150 ng oligo- (dT.) Are added to the RNA in a final volume of 12 μl. The mixture is heated at 70 ^° C. for 10 minutes and then immediately cooled on ice. Then 4 μl of the 5X first strand buffer, 2 μl of 0. IM DTT, 1 μl of 10 mM dNTP mix (in each case 10 mM dATP, dCTP, dGTP and dTTP) and RNase inhibitor (5 units, Böhringer Mannheim) are added. The mixture is heated to 42 ^° C. for 2 minutes, RT 'Superscript ™ II enzyme (300 units, Life Technologies) is added and incubated at 42 ^° C. for 50 minutes. A 35-base oligonucleotide with 17 dT residues and an adapter sequence is used as the oligo (dT) primer (Frohman MA (1990) Acade ic Press, San Diego, CA. pp. 28-38):

5'-GGGAATTCGTCGACAAGC (T) ι ₇ -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 approach is composed as follows:

2.5 μg first strand cDNA

IX PCR Buffer II Perkin Elmer (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 Elmer) dest. 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 with 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 using 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 is carried out as specified in the manufacturer's protocol of the Amersham company. The standardization of the method is verified using primers specifically 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-MYBll:

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 above conditions. (EMBL AF062863; Kranz et al., 1998). The result of the RT-PCR analysis is shown in FIG. 6. Example 3: Analysis of Atmybll Expression by in situ hybridization

Flowers and pods are collected at different stages of development from 7-week-old plants grown on soil as described above. The samples are immediately fixed in freshly prepared 4% p-formaldehyde solution in PBS buffer (130 mM NaCl, 7 mM NaHP0 ₄ , 3 mM NaH ₂ P0 ₄ ) under vacuum for 3 hours. The fixed material is stored in 70% ethanol at 4 ° C until further use. Fixation and embedding work is 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 described step has been shortened to 1 hour. The template for the in-si tu hybridization sample is prepared as described in the instructions for the Lig'nScribe ™ PCR Promoter Addition Kit (Ambion, USA). For this purpose, 20 ng of the PCR fragment, which is obtained with the primers Z17F2 and Z17R3, starting from a partial cDNA clone of the AtMYBll gene (EMBL AF062863; Kranz et al., 1998 (see above), against a reading direction behind a T7 promoter adapter cloned (Ambion, USA)

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 antisense strand of the AtMYBll 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, 2 μl of the ligation reaction and the primers Z17F2 and the PCR adapter primer 1

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 AtMYBll probe. The PCR conditions for producing the templates for the antisense and sense strands of the AtMYBll probes are identical and are carried out under the conditions described in Example 2. In deviation 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 Röche Diagnostics: The following components are combined in one RNase-free Eppendorf tube, which is kept on ice, mixed in the order shown. The final concentration in a reaction volume of 20μl is given. 0.1 μg ethanol-precipitated PCR product, lx labeling mixture (as lOx Mix with 10MM ATP, 10MM CTP, 10MM GTP; 6.5mM UTP, 3.5mM DIG-UTP in Tris-HCl, pH 7.5), lx transcription buffer (as lOx buffer with 400mM Tris-HCl, pH 8.0, 60mM MgCl _{2 /} lOOmM dithioerythritol, 20mM spermidine, lOOmM NaCl, 1 unit / ml RNase inhibitor) and 40 5 units T7 RNA polymerase. The mixture is shaken briefly and centrifuged for a few seconds. The mixture is then incubated at 37 ² C for at least 2 hours. To digest the DNA-PCR product, 20 units of DNase are added and incubated at 37 ^S C for 15 min. 10 2 μl of the supplied EDTA solution are added to terminate the reaction.

8 μm-thick sections of the fixed plant material are cut through a microtome using type S35 microtome blades (Fether, Japan) (Minot microtome type 1212; Leitz Wetzlar,

15 Germany), applied to poly-L-lysine-loaded slides and treated as in Coen et al. (Coen ES et al., Cell 1990, 63: 1311-1322). Before hybridization, the sections are placed in xylene to remove the paraffin and then rehydrated with a series of ethanol dilutions.

20 years. 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. The mixture is then acetylated with acetic anhydride, dehydrated with a series of dilutions of ethanol and air-dried. The DIG-11-UTP labeled RNA probes undergo mild alkaline hydrolysis

25 with warming to 60 ° C for one hour in 100mM carbonate buffer (pH 10.2) to achieve an average fragment length of approximately 150 bases.

Approximately 20 ng of the DIG-11 UTP labeled RNA probe in each

30 50 μl hybridization buffer preheated to 70 ° C. (50% formamide, 300 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, IX Denhardts, 10% dextran sulfate, 10 mM DTT, 250 ng / l tRNA, 100 μg / ml polyA) are used for each slide and incubated with the tissue sample at 45 ° C. overnight. The carriers are 45 min in 2X SSC

35 (20X SSC: 0.3M sodium citrate, 3M 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 washed with 20 μg / ml RNase A in NTE (0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA) treated at 37 ° C for 30 min. The carriers are then again 15 min in 2X SSC at room

Washed 40 temperature and 15 min in 2X SSC at 50 ° C. The immunological detection of the probe is carried out according to the instructions of the "digoxigenin-nucleic acid detection kit" (Röche Diagnostics): For this, the carriers are gently agitated for 1 hour in 1% blocking solution (Röche Diagnostics) in 100 M Tris-HCl

45 (pH 7.5), 150 mM NaCl and then incubated for 30 min in buffer A (0.5% bovine serum albumin, 0.3% Triton-XIOO, 100 mM Tris-HCl (pH 7.5), 150 mM NaCl). Then the carrier with the Antibody conjugate in a 1: 1500 dilution in buffer A and washed three times for 20 min in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl. The supports 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 Incubate -4-chloro-3-indolylphosphate toluidinium salt in 100mM Tris-HCl (pH 9.5), 100mM NaCl and 50mM MgCl ₂ . The color reaction is stopped with 10 mM Tris-HCl (pH 8), ImM 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 fixed with Euparal (BDH). The tissue is then documented under a suitable microscope (eg Leica DMRB) with a camera.

Example 4: Cloning of the AtMYBll promoter

To isolate the full AtMYBll promoter, genomic ^'DNA from Arabidopsis thaliana (ecotype Columbia) is extracted 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:

80ng Genomic DNA I X Expand ™ Long Template PCR Buffer 1

2.5 mM MgCl ₂ , each 350 μM of dATP, dCTP, dGTP and dTTP each 300 nM of each primer - (SEQ ID NO: 11 and 12)

2.5 Units Expand ™ Long Template Poly erase (Röche 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-T0P0 (Invitrogen) according to the manufacturer's instructions, ie the PCR product obtained is inserted into a vector with T overhangs using its A overhangs and a topoisomerase. The construct obtained is 5 pCR4-T0P0-mybll (Fig. 7, I).

Example 5: Deletion analysis

The method is described by Rouster (Rouster J et al., Plant Journal 10 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 mybll promoter, i.e. The 15 nucleic acid sections relevant to its specificity are narrowed by producing different reporter gene expression vectors which contain the entire promoter region on the one hand and various fragments thereof on the other hand. On the one hand, the entire promoter region or fragments thereof are cloned into the pGPTV-GUS-Kan 0 vector (Becker D et al., 1992 Plant Mol Biol 20: 1195-1197). On the one hand, fragments are used for this, which are obtained by using restriction enzymes for the internal restriction sites in the full-length promoter sequence. On the other hand, PCR fragments are used which are provided with interfaces introduced by primers.

For the cloning of the full-length promoter, the vector pCR4-T0P0-mybll is digested with Hindlll and Smal and the fragment isolated from a 1% agarose gel is cloned into the pGPTV-GUS channel. The construct obtained is designated pGPTV-mybll:: GUS (Fig. 7, construct II).

For a first truncated promoter, pCR4-T0P0-mybll is digested with EcoRI and the 5 ca.2050 base pairs fragment isolated from a 1% agarose gel is subcloned into pBluescript SK (Stratagene). The insert is obtained from the resulting vector pBSK-ybll (FIG. 9, construct IX) by digestion with HindII and Smal, isolated over a 1% agarose gel and cloned into pGPTV-GUS-Kan. The construct obtained bears the designation 0 pGPTVl-mybll :: GUS (FIG. 7, construct III).

For a second shortened promoter, pBSK-mybll is digested with EcoRV and Ncol. Overhanging ends are digested with T4 DNA polymerase and the plasmid is religated with blunt ends (as described in Sambrook et al., 5, Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, 1989) to pBM-ybll ( Fig. 9, construct X). The Ver- The shortened insert is obtained from the vector pBM-mybll by digestion with Hinllll and Smal, isolated using a 1% agarose gel and cloned into pGPTV-GUS-Kan. The construct obtained is called pGPTV2-mybll :: GUS (Fig. 7, construct IV).

For a third shortened promoter, pCR4-T0P0-mybll is used as a template for a PCR. The Hindlll and Smal interfaces are introduced using the following primers

Forward primer 5 '-AAGCTTAACCAATCAGGATTAAG-3' (SEQ JD O: 13) and

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

The fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan. The construct obtained is called pGPTV3-mybll :: GUS (Fig. 8, construct V).

For a fourth shortened promoter, pCR4-T0P0-mybll is used as a template for a PCR. The Hindlll and Sall interfaces are introduced using the following primers

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

Reverse primer 5 '-GTCGACAAAATCACTCACTTCACT-3' (SEQ ID O: 4).

The fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan. The construct obtained bears the designation ^• GPTV4-mybll:: GUS-Kan (FIG. 8, construct VI).

For a fifth shortened promoter, pCR4-T0P0-mybll is used as a template for a PCR. The Hindlll and Xbal interfaces are introduced using the following primers

Forward primer 5 '-AAGCTTTGGACAATCATGTCATA-3' _. (SEQ ID O: 5) and

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

The fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan. The construct obtained is called pGPTV5-mybll:: GUS-Kan (Fig. 8, construct VII).

For a sixth shortened promoter, pCR4-T0P0-mybll is used as a template for a PCR. The Hindlll and Xbal interfaces are introduced using 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 again cloned in pGPTV-GUS-Kan. The construct obtained is called pGPTV6-mybll:: GUS-Kan (Fig. 8, construct VIII).

Example 6: Transformation and regeneration of transgenic Arabidopsis thaliana (Columbia) plants

To produce transgenic Arabidopsis plants, i-grrojbacterium tumefaciens (strain C58C1 pGV2260) is transformed with various mybll promoter-GUS vector constructs. The agrobacterial strains are then used to produce transgenic plants. For this purpose, a single transformed agrobacterium colony in a 4 ml culture (medium: YEB medium with 50 μg / ml

Kanamycin and 25 μg / ml rifampicin) incubated overnight at 28 ° C. A 400 ml culture is then inoculated with this culture in the same medium, incubated overnight (28 ° C., 220 rpm) and centrifuged off (GSA rotor, 8,000 rpm, 20 min). The pellet is resuspended in infiltration medium (.1 / 2 MS medium; 0.5 g / l MES, pH 5.8; 50 g / 1 sucrose). The suspension is placed in a plant box (Duchefa) and 100 ml of SILVET L-77 (heptamethyltrisiloxane modified with polyalkylene oxide; Osi Specialties Inc., Cat. P030196) was added to a final concentration of 0.02%. The plant box with 8 to 12 plants is exposed to a vacuum in a desicator for 10 to 15 minutes, followed by spontaneous ventilation. This is repeated 2 to 3 times. Then all plants are planted in plant pots with moist earth and grown under long day conditions (temperature 22 to 24 ° C, night temperature 19 ° C; 65% relative air humidity). After 6 weeks, the seeds are harvested.

Alternatively, transgenic Arabidopsis plants can be obtained by root transformation. White root sprouts from plants up to 8 weeks old are used. To do this, plants are placed under sterile conditions in 1 MS medium (1% sucrose; 100mg / l inositol; l, 0mg / l thiamine; 0.5 mg / 1 pyridoxine; 0.5 mg / 1 - nicotinic acid; 0.5 g MES, pH 5, 7; 0.8% agar) are used. Roots are grown on kailus-inducing medium for 3 days (Ix Gambourg's B5 medium; 2% glucose; 0.5 g / 1

Mercaptoethanol; 0.8% agar; 0.5 mg / 1 2, 4-D (2, 4-dichlorophenoxyacetic acid); 0.05 mg / 1 kinetin). Root sections 0.5 cm long are transferred to 10 to 20 ml callus-inducing liquid medium (composition as described above, but without agar addition) and with 1 ml of the overnight agrobacterial culture described above (grown at 28 ° C., 200 rpm in LB ) inoculated and shaken for 2 min. The root explants are removed after removing excess transferring the liquid medium by dripping into callus-inducing medium with agar, then incubating in callus-inducing liquid medium without agar (with 500 mg / l betabactyl, SmithKline Beecham Pharma GmbH, Munich) with shaking and finally in sprout-inducing medium (5 mg / 1 2-isopentenyl adenine phosphate; 0.15 mg / 1 indole-3-acetic acid; 50 mg / 1 kanamycin; 500 mg / l betabactyl). After 5 weeks and 1 or 2 changes of medium, the small, green sprouts are placed on germination medium (1 MS medium; 1% sucrose; 100 mg / 1 inositol; 1.0 mg / l thiamine; 0.5 mg / 1 pyridoxine; 0.5 mg / 1 nicotinic acid; 0.5 g MES, pH 5, 7; 0.8% agar) and regenerated into plants.

Example 7: Detection of tissue-specific expression

In order to determine the properties of the promoter and to identify the essential elements of it, which make up its tissue specificity, it is necessary to place the promoter itself and various fragments thereof in front of a so-called reporter gene, which enables expression activity to be determined. The bacterial β-glucuronidase may be mentioned as an example (Jefferson et al., EMBO J. 1987, 6, 3901-3907). The ß-glucuroni-. this activity can be determined in-planta using a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405). For the tissue specificity studies, the plant tissue is cut, embedded, stained and analyzed as described (e.g. Bäumlein H et al., 1991 Mol Gen Genet 225: 121-128).

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

Claims

claims 1. Containing an expression cassette for the transgenic expression of nucleic acids a) the promoter of the mybll gene from Arabidopsis thaliana according to SEQ ID NO: 1, or b) functional equivalents or equivalent fragments of a) which have essentially the same promoter activities as a), where 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 sequence to be expressed a) the expression of a protein encoded by said nucleic acid sequence, or b) enables expression of a sense or anti-sense RNA encoded by said nucleic acid sequence. Sign.- 4. Expression cassette according to claims 1 to 3, wherein the nucleic acid sequence to be expressed transgenically is selected from nucleic acids coding for amino acid transporters, saccharide transporters, chalcone synthases, photolyases, photoreceptors, phytases, lipases, triacylglycerol lipases, acetyl-CoA carboxylases, endochitinases , the N-hydroxylating, multifunctional cytochrome P-450, the transcriptional activator CBF1, invertases, the 2S albumin from Bertholletia ex-celsa, endoxyl.glucan transferases, 'phytoendesaturases, the ABI3 protein, the VP-1 protein, "antifreeze "Proteins, glutamate dehydrogenases, a late embryogenetic gene (LEA), Cal. cium-dependent protein kinases, calcineurins, farnesyl transferases, ferritin, oxalate oxidase, DREBlA factor, trehalose phosphate synthase, trehal sephosphate phosphatase, trehalase, cellulases, chitinases, glucanases, ribosome inactivators Proteins, lysozymes, Bacillus thuringiensis endotoxin, a-amylase inhibitor, protease inhibitors, lectins, RMAases, ribozymes, fatty acid desaturases, fatty acid elongases, caffeine acid-O-methyltransferase, cinnamoyl alcohol dehydrogenase, ANT gene. 5. An expression cassette according to claims 1 to ^'4, wherein the transgene is selected to be expressed nucleic acid sequence from the group of nucleic acid sequences with the GenBank accession numbers X92657, M20308, .AJ002399, BAB00748, A19451, U62549, U4Ö256, AB039325, L25042, S78423 , U32624, U77378, V01311, AB044391, AF163819, X78815, AF306348, M60214, X68141, AJ222980, AF078796.- 6. Vectors containing an expression cassette according to claims 1 to 5. 7. Transgenic organism transformed with an expression cassette according to claims 1 to 5 or a vector according to claim 6. 8. Transgenic organism according to claim 7 selected from the group consisting of bacteria, yeasts, fungi, animal and plant organisms 9. cell cultures, parts or transgenic reproductive material derived from a transgenic organism according to claims 7 or 8. 10. Use of a transgenic organism according to one of claims 7 to 8 or cell cultures derived therefrom, parts or transgenic propagation material according to claim 9 for the manufacture processing of food, feed, seeds, pharmaceuticals or fine chemicals. 11. A process for the production of pharmaceuticals or fine chemicals in transgenic organisms according to one of claims 7 or 8 or derived from these cell cultures, parts or transgenic material according to claim 9, characterized in that the transgenic organism is grown and the desired pharmaceutical or the desired fine chemical is isolated. 12. The method according to claim 11, wherein the fine chemicals are enzymes, vitamins, amino acids, sugars, saturated or unsaturated fatty acids, natural or synthetic flavors, flavorings or colorants.