DE10133407A1 - New expression cassette for transgene expression, useful particularly in selection of transformed plants, contain specific constitutive Arabidopsis promoters - Google Patents

Abstract

Expression cassette (EC) for transgenic expression of nucleic acid (NA) comprises, functionally linked to NA, any of the promoters (S1; 836 bp); (S2; 635 bp) or (S3; 2038 bp), or their functional equivalents or functional fragments with practically the same promoter activity. All sequences are defined in the specification. Independent claims are also included for the following: (1) vector containing EC; (2) method for transgenic expression of NA by linking it to one of the new promoters; (3) method for selecting transformed organisms which contain a selection marker sequence linked to one of the new promoters; (4) transgenic organisms transformed with EC or the vector of (1); (5) cell cultures, parts and transgenic replicative materials from the organisms of (4); and (6) method for producing pharmaceuticals and fine chemicals in transgenic organisms of (4).

Description

The invention relates to expression cassettes and vectors that contain herbal constitutive promoters, as well as the Use of these expression cassettes or vectors for transgenic Expression of nucleic acid sequences - preferably selection markers - in organisms, preferably in plants. The invention relates also with these expression cassettes or vectors transformed transgenic plants, crops derived therefrom, parts or Propagation material, and the use of the same for production of food, feed, seeds, pharmaceuticals or Fine chemicals.

The aim of biotechnological work on plants is to manufacture them of plants with improved properties, for example for Increase agricultural productivity. The Production of transgenic plants is a basic technique of Plant biotechnology and therefore an essential prerequisite for basic plant research, as well as for production of plants with improved new properties for the Agriculture, to improve the quality of food or Production of certain chemicals or pharmaceuticals (Dunwell JM, J Exp Bot. 2000; 51 Spec No: 487-96). Often they are natural Defense mechanisms of the plant, for example, against pathogens insufficient. The introduction of foreign genes from plants, animals or Defense can be enhanced by microbial sources. Examples are the Protection against insect damage in tobacco through expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al. (1987) Nature 328: 33-37) or the protection of tobacco against fungal attack by expression of a chitinase from the bean under the control of the CaMV promoter (Broglie et al. (1991) Science 254: 1194-1197). Furthermore, through the introduction of foreign genes herbicide resistance can be achieved, which is the growing conditions optimized and crop losses reduced (Ott KH et al. (1996) J Mol Biol 263 (2): 359-368). Also, the quality of the products be improved. So the durability and shelf life of Harvest products, for example, by inactivating certain Ripening genes can be increased. This was shown for example the tomato by inactivating the polygalacturonase (Hamilton AJ et al. (1995) Curr Top Microbiol Immunol 197: 77-89).

A basic requirement for the transgenic expression of certain Genes in plants are more plant-specific Promoters. Various plant promoters are known. there can distinguish between constitutive promoters that are local and Expression in different parts limited in time a plant, and specific promoters that a Expression only in certain parts or cells of a plant (e.g. root, seeds, pollen, leaves etc.) or just too allow certain times of development to be distinguished. For example, constitutive promoters are used for expression so-called selection marker used. Selection marker (e.g. Antibiotic or herbicide resistance genes) allow that Transformation event from the multitude of untransformed, but otherwise identical vegetable individuals filter out.

Constitutive, plant promoters have so far been relatively rare written. The TR double promoter or the Promoters of vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991).

The constitutive predominantly currently used in plants Promoters are almost exclusively viral or from Agrobacterium isolated promoters. This is the individual Nopaline synthase (nos) promoter (Shaw et al. (1984) Nucleic Acids Res. 12 (20): 7831-7846), the mannopine synthase (mas) promoter (Comai et al. (1990) Plant Mol Biol 15 (3): 373-381) and the Octopine synthase (ocs) promoter (Leisner and Gelvin (1988) Proc Natl Acad Sci USA 85 (5): 2553-2557) from Agrobacterium tunefaciens or the CaMV35S promoter from the cauliflower mosaic virus. The latter is the most commonly used promoter in expression systems with ubiquitous and persistent expression (Odell et al. (1985) Nature 313: 810-812; Battraw and Hall (1990) Plant Mol Biol 15: 527-538; Benfey et al. (1990) EMBO J 9 (69): 1677-1684; US 5,612,472). Often used as a constitutive promoter However, CaMV 35S promoter shows variations in activity in different plants and in different tissues same plant (Atanassova et al. (1998) Plant Mol Biol 37: 275-85; Battraw and Hall (1990) Plant Mol Biol 15: 527-538; Holtorf et al. (1995) Plant Mol Biol 29: 637-646; Jefferson et al. (1987) EMBO J 6: 3901-3907). Another disadvantage of the 35S Promotors is that in case of infection with the cauliflower mosaic virus and its typical pathogenic variants the expression of Transgene is changed. So are plants that contain the BAR gene control of the 35S promoter no longer express resistant after infection with the virus that is typically found in nature occurs (Al-Kaff et al. (2000) Natur Biotechnology 18: 995-99).

As an alternative to the CaMV 35S promoter from the viral area The Sugarcane bacilliform badnavirus (ScBV) was promoted (Schenk et al. (1999) Plant Mol Biol 39 (6): 1221-1230), which conveys an expression pattern similar to the CamV. The ScBV promoter activity was transient Expression analysis with various dicotyledonous plants, including Nicotiana tabacurn and N. benthamiana, sunflower and rapeseed, and monocotyledonous plants, here using banana, corn and millet, analyzed. The ScBV was in the transient analyzes in maize Expression level mediated by promoter comparable to that of the Corn ubiquitin promoters (see below). Furthermore, the ScBV Promoter-mediated expression rate in transgenic banana and Tobacco plants tested and showed in both plant species in essential constitutive expression.

As common promoters for the expression of selection markers in Plants are primarily the nos promoter, but also the mass and ocs promoter, all of which are used Agrobacterium strains were isolated.

The use of viral sequences is on the part of the consumer often met with great reservations. These concerns will not last nourished by examinations that the safety of the 35S CaMV promoters - due to a possible horizontal Gene transfers due to a recombination "hot spot" - in question (Ho MW et al. (1999) Microbial Ecology in Health and Disease 11: 194-197; Cummins J et al. (2000) Nature Biotechnology 18: 363). A goal of future biotechnological work Plants are therefore the replacement of viral genetic elements by regulatory herbal elements to be as close as possible to herbal system to stay.

Because of the prevailing concerns about viral There are extensive promoters to this through herbal promoters To replace promoters. A comparable one to the viral elements So far, however, there is no promoter of plant origin have been described.

A herbal ubiquitin promoter has been described Arabidopsis thaliana (Callis et al. (1990) J Biol Chem 265: 12,486 to 12,493; Holtorf S et al. (1995) Plant Mol Biol 29: 637-747). Contrary to the findings in the articles mentioned, our own results Studies show that the Arabidopsis ubiquitin promoter for the Expression of selection marker genes is unsuitable and its general applicability are questioned for this reason must (see Comparative Examples 1 and 3).

The expression pattern mediated by the maize ubiquitin promoter has been described for the maize Ubi-1 and Ubi-2 promoters (Christensen et al. (1992) Plant Mol Biol 18 (4): 675-689). While the Ubi-1 promoter is good in maize and other monocotyledonous plants Has expression activity, he shows in dicotyledons Tobacco plants only 10% of the activity in comparable experiments with the 35S viral promoter. Further was demonstrated that the maize Ubi-1 promoter for overexpression of Genes in monocotyledonous plant systems and above is also sufficiently strong to be herbicide resistant through expression of selection markers (Christensen and Quail (1996) Transgenic Res 5 (3): 213-218). For dicotyledons The Ubi-1 promoter proved to be unsuitable for expression systems.

A comparison of the organ specificity and strength of different constitutive promoters was developed by Holtorf (Holtorf et al. (1995) Plant Mol Biol 29 (4): 637-646) using stably transformed Arabidopsis plants performed. The study included a. the CaMV35S promoter, the leaf-specific thionine promoter Barley and the Arabidopsis Ubiquitin Promoter (UBQ1). The highest The CaMV35S promoter showed expression rate. Based on Use of an additional translational enhancer (TMV omega element) the expression rate of the promoter was unchanged Organ specificity can be increased two to three times. The leaf-specific barley thionine promoter was in the majority of the transformed lines become inactive while the UBQ1 promoter is off Arabidopsis showed medium expression rates.

McElroy and co-workers reported one on the rice Actin 1 (Act1) promoter-based construct for transformation monocotyledonous plants (McElroy et al. (1991) Mol Gen Genet 231: 150-160). Overall, was from the previously described Studies concluded that those on the Act1 promoter based expression vectors are suitable, a sufficient strong and constitutive expression of foreign DNA in to control transformed cells of monocotyledonous plants.

The promoter of an S-adenosyl-L-methionine synthetase is also described as a constitutive promoter (WO 00/37662). The main disadvantage here is that the expression level is dependent on the methionine concentration (see WO 00/37662; FIG. 7).

WO 99/31258 describes chimeric, constitutive plant Promoters made up of different elements different Assemble promoters with complementary expression patterns so that the combination of individual tissue specificities additive to one constitutive expression pattern.

The Ferredoxin NADPH Oxidoreductase (FNR) is a protein of the Electron transport chain and reduced NADP + to NADPH. Experiments in spinach with the spinach FNR promoter, which with the GUS gene merged, indicate a light - inducible element in the FNR promoter (Oelmüller et al. (1993) Mol. Gen. Genet. 237: 261-72). Because of its function, one would be for the promoter strictly leaf-specific expression pattern was to be expected. Due to the tissue-dependent expression pattern, this would be Promoter poorly suited for expression of selection markers. Here selection in as many tissue parts as possible is required, to ensure an efficient selection.

The promoter of the triose phosphate translocator (TPT) should mainly due to its function during photosynthesis be leaf-specific. The cDNAs from Kartoffel (Schulz et al. (1993) Mol Gen Genet 238: 357-61), cauliflower (Fischer et al. (1997) Plant Cell 9: 453-62), rapeseed (WO 97/25346) and maize Kammerer B (1998) The Plant Cell 10: 105-117). Kammerer et al. show that the maize TPT mRNA expression is high in the leaves and the stamens. However, there is no expression in the stem or watching the roots. Because of the tissue-dependent Expression pattern, the promoter would be poorly suited for Expression of selection markers. Here is a selection in if possible all tissue parts required for an efficient selection guarantee.

• 1. Mangelhafte Homogenität der Expression:
  Die bekannten, sogenannten "konstitutiven" Promotoren zeigen vielfach eine unterschiedliche Expressionshöhe je nach Gewebe- oder Zelltyp. Zudem ist die Expressionseigenschaft oft stark von dem Insertionsort des Wirtsgenoms abhängig. Dies hat zur Folge, dass die durch heterologe Expression zu erzielenden Effekte nicht in dem gleichen Ausmaß homogen in der Pflanze erreicht werden können. Es kann zu Unter- oder Überdosierungen kommen. Dies kann sich nachteilig auf das Pflanzenwachstum oder den Pflanzenwert auswirken.
• 2. Mangelhaftes Zeitprofil:
  Die im Stand der Technik bekannten sogenannten "konstitutiven" Promotoren zeigen oft eine nicht konsitente Aktivität während der Entwicklung eines Gewebes. Damit können zum Beispiel in der frühen Phase der somatischen Embryogenese keine wünschenswerten Effekte (wie Selektion) erzielt werden, was gerade hier - aufgrund der Empfindlichkeit des Embryos gegen in vitro Bedingungen und Stressfaktoren - vorteilhaft wäre.
• 3. Mangelnde Anwendbarkeit auf viele Pflanzenarten:
  Die im Stand der Technik beschriebenen sogenannten "konstitutiven" Promotoren sind oft nicht in allen Arten in gleicher Weise aktiv.
• 4. Beim Vorliegen mehrerer Expressionskassetten mit jeweils dem gleichen "konstitutiven" Promotor in einem Organismus kann es zu Wechselwirkungen zwischen denselben und sogar zum Abschalten ("Gene Silencing") einzelner Expressionskassetten kommen (Mette et al. (1999) EMBO J. 18: 241-248).
• 5. Promotoren viralen Ursprungs können durch Virusinfektionen der transgenen Pflanze beeinflußt werden und die gewünschte Eigenschaft dann nicht mehr ausprägen (Al-Kaff et al. (2000) Natur Biotechnology 18: 995-99).
• 6. Die öffentliche Akzeptanz gegenüber der Verwendung von Promotoren und Elementen aus pflanzlichen Systemen ist höher als bei viralen Systemen.
• 7. Die Zahl der für die Expression von Selektionsmarkern in Pflanzen geeigneten Promotoren ist gering und sie sind gewöhnlicher Weise viralen oder bakteriellen Ursprungs.
• 8. Pollen/Antheren-Expression: Die genannten Promotoren (wie beispielsweise 35S CaMV) zeigen eine starke Aktivität im Pollen oder den Staubbeuteln (Antheren). Die kann unvorteilhafte Auswirkungen auf die Umwelt haben. So hatte eine unspezifische Expression von Bacillus thuringiensis Endotoxinen nicht nur den gewünschten Effekt auf Fraßinsekten durch Expression in der Wurzel, sondern auch infolge einer Expression im Pollen bedeutende Schäden im Bestand des Monarchfalters zur Folge, der den Pollen als Hauptnahrungsquelle nutzt (Losey JE et al. (1999) Nature 399, 214).

The so-called "constitutive" promoters described in the prior art have one or more of the following disadvantages:

• 1. Poor homogeneity of expression:
  The known, so-called "constitutive" promoters often show a different level of expression depending on the tissue or cell type. In addition, the expression property is often strongly dependent on the insertion site of the host genome. As a result, the effects to be achieved by heterologous expression cannot be achieved to the same extent in the plant to the same extent. Underdosing or overdosing may occur. This can adversely affect plant growth or plant value.
• 2. Poor time profile:
  The so-called "constitutive" promoters known in the prior art often show inconsistent activity during the development of a tissue. In the early phase of somatic embryogenesis, for example, no desirable effects (such as selection) can be achieved with this, which would be advantageous here - due to the sensitivity of the embryo to in vitro conditions and stress factors.
• 3. Inapplicability to many plant species:
  The so-called "constitutive" promoters described in the prior art are often not equally active in all types.
• 4. If there are several expression cassettes with the same “constitutive” promoter in one organism, there may be interactions between them and even gene silencing of individual expression cassettes (Mette et al. (1999) EMBO J. 18: 241 -248).
• 5. Promoters of viral origin can be influenced by virus infections of the transgenic plant and then no longer express the desired property (Al-Kaff et al. (2000) Natur Biotechnology 18: 995-99).
• 6. Public acceptance of the use of promoters and elements from plant systems is higher than that of viral systems.
• 7. The number of promoters suitable for the expression of selection markers in plants is small and they are usually of viral or bacterial origin.
• 8. Pollen / anther expression: The promoters mentioned (such as 35S CaMV) show a strong activity in the pollen or the anthers. This can have adverse effects on the environment. Nonspecific expression of Bacillus thuringiensis endotoxins not only had the desired effect on feeding insects by expression in the root, but also, as a result of expression in pollen, resulted in significant damage to the population of the monarch butterfly, which uses pollen as the main food source (Losey JE et al. (1999) Nature 399, 214).

• a) Möglichst homogene lokale und zeitliche Genexpression d. h. eine Expression in möglichst vielen Zelltypen oder Geweben eines Organismus während der verschiedenen Phasen des Entwicklungszyklus. Gewünscht wird ferner eine effizient Selektion in dedifferentierten Zellen (verschiedenen Kallus-Phasen) aus Gewebekultur und weiteren Entwicklungsstadien, die für die Gewebekultur geeignet sind.
• b) Möglichst breite Anwendbarkeit auf verschiedene Pflanzenarten bzw. Anwendbarkeit auf Arten, in denen mit den bisher bekannten "konstitutiven" Promotoren keine Expression erreicht werden kann.
• c) Um eine Kombination von mehreren Transgenen in einer Pflanze zu realisieren, ist es wünschenswert, mehrere Transformationen hintereinander zu schalten oder Konstrukte mit mehreren Promotor-Kassetten zu verwenden, ohne daß durch die mehrmalige Verwendung identischer regulatorischer Sequenzen Silencing-Effekte erzeugt werden.
• d) Pflanzlichen Ursprung zur Vermeidung von Akzeptanzproblemen beim Konsumenten und eventuellen zukünftigen Zulassungsproblemen.
• e) Nebenaktivitäten eines Promotors in den Antheren/Pollen sind nicht wünschenswert, zum Beispiel um Umweltschäden zu vermeiden (s. o.).

An ideal constitutive promoter should have as many of the following properties as possible:

• a) As homogeneous as possible local and temporal gene expression ie an expression in as many cell types or tissues of an organism as possible during the different phases of the development cycle. What is also desired is an efficient selection in dedifferentiated cells (different callus phases) from tissue culture and further development stages which are suitable for tissue culture.
• b) The broadest possible applicability to different plant species or applicability to species in which no expression can be achieved with the "constitutive" promoters known to date.
• c) In order to realize a combination of several transgenes in one plant, it is desirable to connect several transformations in series or to use constructs with several promoter cassettes without the silencing effects being generated by the repeated use of identical regulatory sequences.
• d) Vegetable origin to avoid consumer acceptance problems and possible future approval problems.
• e) Secondary activities of a promoter in the anthers / pollen are not desirable, for example to avoid environmental damage (see above).

The object underlying the present invention was so in the provision of herbal regulatory Sequences that have as many of the above properties as possible fulfill, especially a ubiquitous and development-independent (constitutive) expression of an expression to be expressed Nucleic acid sequence - preferably coding for a selection marker convey. Despite various plant promoters for one constitutive expression claimed at least in individual species has so far been no promoter with the above Desired properties described. It therefore existed Task to identify appropriate promoters.

This task was accomplished by providing expression cassettes based on the putative ferredoxin gene promoter (as a result of pFD "putative ferredoxin") gene from Arabidopsis thaliana, of the ferredoxin NADPH oxidoreductase (as a result of FNR) gene Arabidopsis thaliana and the Triose-Phosphate Translocator (TPT) gene from Arabidopsis thaliana:

1. Ferredoxin (FD) promoter from Arabidopsis thaliana

When analyzing the Arabidopsis genome, a putative ORF of a ferredoxin gene identified. The isolated 836 bp 5 'flanked sequence fused to the gene of Surprisingly, glucuronidase showed up in transgenic tobacco constitutive expression pattern. The sequence corresponds to one Sequence section on chromosome 4 of Arabidopsis thaliana as he at GenBank under Acc.-No. Z97337 is deposited (Version Z97337.2; base pair 85117 to 85952; the gene starting from bp 85953 is with "strong similarity to ferredoxin [2Fe-2S] I, Nostoc muscorum "annotated). (The gene is not to be confused with that under GenBank Acc.-No: X51370 annotated A.thaliana gene for preferredoxin; Vorst O et al. (1990) Plant Mol Biol 14 (4): 491-499).

In the anthers / pollen of the closed flower buds could only a weak activity in which the ripe flowers even no more can be detected. Contrary to that from the Reservations derived from literature findings against the suitability of the Promoters for the effective expression of selection markers (for Example based on the suspected leaf specificity or the Function in photosynthetic electron transport) a highly efficient selection by combination with for example the phosphinothricin resistance gene (bar / pat) be demonstrated.

2. Ferredoxin NADPH oxidoreductase (FNR) promoter Arabidopsis thaliana

Based on the information, a cDNA from N. tabacum was in the Arabidopis database for a homologous gene searched. According to this sequence information, primers became synthesized. The via PCR from genomic DNA from Arabidopsis thaliana amplified promoter (634 bp), one of which leaf-specific expression was expected, showed in transgenic tobacco plants a surprisingly ubiquitous and of Expression independent of insertion site.

The promoter sequence corresponds in part to one Sequence section on chromosome 5 of Arabidopsis thaliana as described in the GenBank under Acc.-No. AB011474 is deposited (Version AB011474.1 from December 27, 2000; base pair 70127 to 69583; the gene starting from bp 69492 is with "ferredoxin-NADP + reductase "annotated).

No activity could be detected in the anthers / pollen become. Contrary to what is derived from the literature findings Reserved against suitability of the promoter for effective Expression of selection markers (for example due to the suspected leaf specificity or function in photosynthetic electron transport), could be a highly efficient Selection by combination with, for example, the Phosphinothricin resistance gene (bar / pat) will be demonstrated.

3. Arabidopsis triose-phosphate translocator (TPT) promoter thaliana

A 2041bp PCR fragment was amplified starting of Arabidopsis gene bank data from chromosome V, clone MCL19.10. The promoter sequence corresponds to one Sequence section on chromosome 5 of Arabidopsis thaliana as described in the GenBank under Acc.-No. AB006698 is deposited (Version AB006698.1 from December 27, 2000; base pair 53242 to 55281; the gene starting with bp 55282 is with "phosphate / triose-phosphate translocator" annotated).

Surprisingly, transgenic tobacco plants did not show only a strong activity in numerous parts of plants. In no activity could be detected in the anthers / pollen. Contrary to what is derived from the literature findings Reserved against suitability of the promoter for effective Expression of selection markers (for example due to the suspected leaf specificity), could make a highly efficient selection by combination with, for example, the Phosphinothricin resistance gene (bar / pat) will be demonstrated.

The ubiquitous expression pattern, but above all that Ability of the TPT promoter to express Selection markers are a big surprise for the Specialist because the triose phosphate translocator is responsible for the exchange of C3 sugar phosphates between cytosol and plastids in photosynthetic sheets. The TPT is localized in the inner chloroplast membrane. Colorless Plastids typically contain hexose transporters the C6 sugar phosphate exchange takes place. It is not too expect such genes in the early callus and Embryogenesis stages are active (Stitt (1997) Plant Metabolism, 2nd ed., Dennis eds. Longman Press, Harlow, UK, 382-400).

Both the pFD, FNR and the TPT promoter were found to be sufficiently strong to be particularly nucleic acid sequences Successfully express selection marker genes.

Furthermore, the Arabidopsis thaliana ubiquitin promoter (Holtorf et al. (1995) Plant Mol Biol 29: 637-646) and the squalene synthase promoter (Kribii et al. (1997) Eur J Biochem 249: 61-69), but both were surprisingly not suitable To mediate selection marker gene expression, although the literature data of Ubiquitin promoters from monocots (see above) had suggested that especially the ubiquitin promoter of a dicotyledonous plant A promoter of a selection marker system should have worked (see comparative examples 1 and 3). The same applies to the Squalene synthase promoter, the characterization of which would have been expected let that sufficiently high expression rates for the successful control of a selection marker gene can be achieved (Del Arco and Boronat (1999) 4th European Symposium on Plant Isoprenoids, April 21-23, 1999, Barcelona, Spain) (see Comparative Examples 2 and 3).

• a) einen Promotor gemäss SEQ ID NO: 1, 2 oder 3,
• b) ein funktionelles Äquivalent oder äquivalentes Fragment von a), das im wesentlichen die gleiche Promotoraktivität wie a) besitzt,

wobei a) oder b) funktionell mit einer transgen zu exprimierenden Nukleinsäuresequenz verknüpft sind. A first subject of the invention therefore relates to expression cassettes for the transgenic expression of nucleic acids comprising

• a) a promoter according to SEQ ID NO: 1, 2 or 3,
• b) a functional equivalent or equivalent fragment of a) which has essentially the same promoter activity as a),

where a) or b) are functionally linked to a transgenic nucleic acid sequence.

• a) einen Promotor gemäss SEQ ID NO: 1, 2 oder 3, oder
• b) einem funktionelle Äquivalent oder äquivalente Fragment von a), das im wesentlichen die gleichen Promotoraktivitäten wie a) besitzt,

transgen exprimiert wird. The invention further relates to methods for the transgenic expression of nucleic acids, characterized in that a nucleic acid sequence is functionally linked to

• a) a promoter according to SEQ ID NO: 1, 2 or 3, or
• b) a functional equivalent or equivalent fragment of a) which has essentially the same promoter activities as a),

is expressed transgenically.

Expression includes transcription of the transgene expressing nucleic acid sequence, but can - in the case of an open Reading frames in "sense" orientation - also the translation of the transcribed RNA of the nucleic acid sequence to be expressed transgenically into a corresponding polypeptide.

• a) ein Promotor gemäss SEQ ID NO: 1, 2 oder 3 oder ein funktionelles Äquivalent oder äquivalente Fragment derselben, oder
• b) die zu exprimierende Nukleinsäuresequenz, oder
• c) (a) und (b)

sich nicht in ihrer natürlichen, genetischen Umgebung (d. h. an ihrem natürlichen chromosomalen Locus) befinden oder durch gentechnische Methoden modifiziert wurden, wobei die Modifikation beispielhaft eine Substitution, Addition, Deletion, Inversion oder Insertion eines oder mehrerer Nukleotidreste sein kann. Expression cassette for the transgenic expression of nucleic acids or methods for the transgenic expression of nucleic acids encompass all such constructions or methods which have been brought about by genetic engineering methods and in which either

• a) a promoter according to SEQ ID NO: 1, 2 or 3 or a functional equivalent or equivalent fragment thereof, or
• b) the nucleic acid sequence to be expressed, or
• c) (a) and (b)

are not in their natural, genetic environment (ie 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.

The expression cassettes according to the invention, by them derived vectors or the methods according to the invention can functional equivalents to those under SEQ ID NO: 1, 2 or 3 described promoter sequences include. Functionally equivalent Sequences also include all of the sequences made by the complementary counter strand that defined by SEQ ID NO: 1, 2 or 3 Sequences are derived and essentially the same Have promoter activity.

Functional equivalents with respect to those of the invention Promoter means in particular natural or artificial mutations of the promoter sequences described under SEQ ID NO: 1, 2 or 3 as well as its homologues from other plant genera and species, which continues to have essentially the same promoter activity exhibit.

Promoter activity is considered essentially the same denotes when the transcription of a particular to be expressed Gene under the control of a particular one of SEQ ID NO: 1, 2 or 3 derived promoters under otherwise unchanged conditions has a localization within the plant that is too at least 50%, preferably at least 70%, particularly preferred at least 90% very particularly preferably at least 95% congruent is obtained using a comparison expression one of the promoters described by SEQ ID NO: 1, 2 or 3. The expression level can go down as well as down above compared to a comparison value. Prefers such sequences, their level of expression, are measured on the basis of the transcribed mRNA or the one translated as a result Protein, quantitatively under otherwise unchanged conditions not more than 50%, preferably 25%, particularly preferably 10% of a comparison value obtained with one by SEQ ID NO: 1, 2 or 3 promoter differs. Particularly preferred are those sequences, the expression level of which is measured using the transcribed mRNA or the protein translated as a result, under otherwise unchanged conditions, quantitatively by more than 50%, preferably 100%, particularly preferably 500%, very particularly preferably 1000% receive a comparison value with that obtained from SEQ ID NO: 1, 2 or 3 described promoter exceeds. Is preferred the level of expression of the natural mRNA of the respective gene or the natural gene product. Is preferred furthermore obtained the expression level with a as a comparison value any, but certain nucleic acid sequence, preferred those nucleic acid sequences that are easy to quantify Encode proteins. Are particularly preferred Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as the "green fluorescence protein" (GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23 (5): 912-8, 1997) chloramphenicol transferase, a Luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414) or the β-galactosidase, which is very particularly preferred β-glucuronidase (Jefferson et al. (1987) EMBO J. 6: 3901-3907).

Otherwise unchanged conditions means that the expression, through one of the expression cassettes to be compared is initiated, not by combination with additional ones genetic control sequences, for example enhancer sequences, is modified. Unchanged conditions also means that all Framework conditions such as plant species, Stage of development of the plants, growing conditions, assay conditions (such as Buffers, temperature, substrates etc.) between those to be compared Expressions are kept identical.

Mutations include substitutions, additions, deletions, Inversion or insertions of one or more nucleotide residues. Such nucleotide sequences are thus also carried out, for example the present invention encompasses, which one by Modification of a promoter according to SEQ ID NO: 1, 2 or 3. aim Such a modification can further narrow the scope of the contained sequence or z. B. also the insertion of more Restriction enzyme interfaces, the removal of superfluous DNA or adding more sequences, for example more regulatory sequences.

Where insertions, deletions or substitutions, such as. B. Transitions and transversions, in themselves, can be considered known techniques, such as in vitro mutagenesis, "primer repair", Restriction or ligation can be used. Through manipulations, such as B. restriction, "chewing-back" or replenishment of Overhangs for "blunt ends" can be complementary ends of the fragments for the ligation will be made available. Too analog 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 entire entire length of the sequence, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the following parameters is calculated:
Gap Weight: 12;
Length Weight: 4;
Average match: 2,912;
Average mismatch: -2.003.

An example is a sequence that has a homology of at least 50% nucleic acid based with the sequence SEQ ID NO: 1, 2 or 3, understood a sequence that in a Comparison with the sequence SEQ ID NO. 1, 2 or 3 according to the above Program algorithm with the above parameter set a homology of has at least 50%.

Functional homologs to the above promoters Use in the expression cassettes according to the invention comprises prefers those sequences which have a homology of at least 50%, preferably 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, most preferably 99% over a length of at least 100 base pairs, preferably at least 200 base pairs, particularly preferably of at least 300 base pairs, whole particularly preferred of at least 400 base pairs, most often preferably of at least 500 base pairs.

Further examples of the in the invention Expression cassettes or vectors used promoter sequences can be, for example, from different organisms, their genomic sequence is known, such as from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthium anuus, Linum sativum by comparing homology easy to find from databases.

Functional equivalents also means DNA sequences that are under Standard conditions with the nucleic acid sequence coding for a promoter according to SEQ ID NO: 1, 2 or 3 or to it hybridize complementary nucleic acid sequences and the im have essentially the same properties. Standard hybridization conditions is to be understood broadly and means stringent as well less stringent hybridization conditions. Such Hybridization conditions are among others at Sambrook J, Fritsch EF, Maniatis T et al., In Molecular Cloning (A Laboratory Manual), 2. 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 be selected from the range of conditions limited by low stringency (approximately 2X SSC at 50 ° C) and those with high stringency (with about 0.2X SSC at 50 ° C preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition, the temperature during the washing step of low stringent conditions at room temperature, around 22 ° C, raised to more stringent conditions at around 65 ° C become. Both parameters, salt concentration and temperature, can can be varied simultaneously, also one of the two Parameters kept constant and only the other can be varied. While hybridization can also denaturing agents such as Example formamide or SDS can be used. In the presence of 50% formamide, hybridization is preferred at 42 ° C executed. Some exemplary conditions for hybridization and Wash step are given as a result:

• a) 4X SSC bei 65°C, oder
• b) 6X SSC, 0.5% SDS, 100 µg/ml denaturierter, fragmentierte Lachssperma-DNA bei 65°C, oder
• c) 4X SSC, 50% Formamid, bei 42°C, oder
• d) 6X SSC, 0.5% SDS, 100 µg/ml denaturierter, fragmentierte Lachssperma-DNA, 50% Formamid bei 42°C, oder
• e) 2X oder 4X SSC bei 50°C (schwach stringente Bedingung), oder
• f) 2X oder 4X SSC, 30 bis 40% Formamid bei 42°C (schwach stringente Bedingung).
• g) 6x SSC bei 45°C, oder,
• h) 50% Formamid, 4xSSC bei 42°C, oder
• i) 50% (vol/vol) Formamid, 0,1% Rinderserumalbumin, 0,1% Ficoll, 0,1% Polyviylpyrrolidon, 50 mM Natriumphosphatpuffer pH 6,5, 750 mM NaCl, 75mM Natriumcitrate bei 42°C, oder
• j) 0,05 M Natriumphosphatpuffer pH 7,0, 2mM EDTA, 1% BSA und 7% SDS.

(1) Hybridization conditions with, for example

• a) 4X SSC at 65 ° C, or
• b) 6X SSC, 0.5% SDS, 100 µg / ml denatured, fragmented salmon sperm DNA at 65 ° C, or
• c) 4X SSC, 50% formamide, at 42 ° C, or
• d) 6X SSC, 0.5% SDS, 100 µg / ml denatured, fragmented salmon sperm DNA, 50% formamide at 42 ° C, or
• e) 2X or 4X SSC at 50 ° C (weakly stringent condition), or
• f) 2X or 4X SSC, 30 to 40% formamide at 42 ° C (weakly stringent condition).
• g) 6x SSC at 45 ° C, or,
• h) 50% formamide, 4xSSC at 42 ° C, or
• i) 50% (vol / vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42 ° C, or
• j) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and 7% SDS.

(2) washing steps with for example

• a) 0.1X SSC at 65 ° C, or
• b) 0.1X SSC, 0.5% SDS at 68 ° C, or
• c) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C, or
• d) 0.2X SSC, 0.1% SDS at 42 ° C, or
• e) 2X SSC at 65 ° C (weakly stringent condition), or
• f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.

Process for the production of functional Equivalents preferably include the introduction of mutations Promoter according to SEQ ID NO: 1, 2 or 3. Mutagenesis can undirected ("random"), the mutagenized sequences then with regard to their properties after a "trial-by- error "procedure. Particularly advantageous Selection criteria include, for example, increased resistance versus a selection marker, the amount of the resulting Expression of the introduced nucleic acid sequence.

Alternatively, non-essential sequences can be one of the promoters according to the invention are deleted without the aforementioned To significantly impair properties. such Deletion variants represent functionally equivalent parts of the promoter described by SEQ ID NO: 1, 2 or 3 such a deletion mutant the truncated pFD promoter sequence (pFDs) according to SEQ ID NO: 4, which are functional equivalent part is expressly included.

Narrowing the promoter sequence to certain essential ones regulatory regions can also be used to search with the help of search routine be made by promoter elements. Often in those for Promoter activity relevant regions certain promoter elements abundant. This analysis can be done with, for example Computer programs such as the program PLACE ("Plant Cis-acting Regulatory DNA Elements ") (K. Higo et al., (1999) Nucleic Acids Research 27: 1, 297-300) or the BIOBASE database "Transfac" (Biological Databases GmbH, Braunschweig).

Methods for mutagenizing nucleic acid sequences are Known to those skilled in the art and include, for example, the use of Oligonucleotides with one or more mutations compared to the region to be mutated (e.g. as part of a "Site-specific mutagenesis"). Typically, primers come with about 15 up to about 75 nucleotides or more, wherein preferably about 10 to about 25 or more nucleotide residues on both sides the sequence to be changed are localized. Details and Implementation of said mutagenesis methods are familiar to the person skilled in the art (Kunkel et al., Methods Enzymol, 154: 367-382, 1987; Tomic et al. (1990) Nucl Acids Res 12: 1656; Upender, Raj, Weir (1995) Biotechniques 18 (1): 29-30; US 4,237,224). Mutagenesis can also by treating, for example, vectors that are one of the contain nucleic acid sequences according to the invention, with mutagenizing agents such as hydroxylamine can be realized.

The contained in the expression cassettes according to the invention Nucleic acid sequences which are to be expressed transgenically can be used with other genetic control sequences in addition to one of the promoter according to the invention can be functionally linked.

A functional link is, for example the sequential arrangement of the promoter that is transgenic expressing nucleic acid sequence and possibly other regulatory elements such as a terminator such that each of the regulative elements its function in transgenic expression of the Nucleic acid sequence, depending on the arrangement of the nucleic acid sequences to sense or anti-sense RNA. This is not a direct link in the chemical sense is absolutely necessary. Genetic control sequences, such as Enhancer sequences can also function from more distant positions or even from other DNA molecules to the target sequence exercise. Arrays are preferred in which the transgene is too expressing nucleic acid sequence behind that acting as a promoter Sequence is positioned so that both sequences are covalent are interconnected. The distance between is preferred the promoter sequence and the transgene to be expressed Nucleic acid sequence less than 200 base pairs, particularly preferred less than 100 base pairs, very particularly preferably less than 50 Base pairs.

A functional link can be created using common recombination and cloning techniques are realized, 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) are described. But there can also be more between the two sequences Sequences are positioned, for example, the function of a Linkers with certain restriction enzyme interfaces or one Have signal peptides. The insertion of sequences for Expression of fusion proteins result.

The concept of genetic control sequences is broad understand and mean all those sequences that have an impact on the Established or the function of the inventive Expression cassette. Modify genetic control sequences for example transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the Expression cassettes according to the invention 5 'upstream from the respective transgenic nucleic acid sequence to be expressed one of the promoters according to the invention and 3'-downstream one Terminator sequence as an additional genetic control sequence, as well if necessary, further customary regulatory elements, in each case functionally linked to the transgene to be expressed Nucleic acid sequence.

Genetic control sequences also include other promoters Promoter elements or minimal promoters that the can modify expression-controlling properties. So through genetic control sequences, for example the tissue-specific ones Expression 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).

Further promoters can also functionally function with the expressing nucleic acid sequence linked to expression in other plant tissues or in other organisms, such as Example E.coli enable bacteria. As plant promoters in principle all promoters described above come into question. For example, it is conceivable that a certain Nucleic acid sequence by a promoter (for example one of the promoters according to the invention) in a plant tissue as sense RNA is transcribed and translated into the corresponding protein while using the same nucleic acid sequence through a different promoter another specificity in a different tissue to anti-sense RNA transcribed and the corresponding protein down-regulated becomes. This can be done by an inventive Expression cassette can be realized by the one promoter in front of the transgene the nucleic acid sequence to be expressed is positioned and the other promoter behind it.

Genetic control sequences also include the 5 'untranslated region, introns or the 3' non-coding region of Genes, preferably gene. It has been shown that this one significant functions in the regulation of gene expression can play. So it was shown that 5'-untranslated Sequences that can enhance transient expression of heterologous genes. They can also promote tissue specificity (Rouster J et al. (1998) Plant J. 15: 435-440.). Conversely, suppresses the 5'-untranslated region of the opaque-2 gene expression. A Deletion of the corresponding region leads to an increase in Gene activity (Lohmer S et al. (1993) Plant Cell 5: 65-73). The below SEQ ID NO: 1, 2 or 3 specified nucleic acid sequence contains the Section gene that promoter and 5'-untranslated Region up to the ATG start codon protein represented.

McElroy et al. (McElroy et al. (1991) Mol Gen Genet 231 (1): 150-160) reported one on the rice Actin 1 (Act1) Promoter-based construct for the transformation of monocotyledons Plants. In transgenic rice cells, the use of the Act1 introns in combination with the 35S promoter into one compared to the isolated 35S promoter increased tenfold Expression rate. An optimization of the sequence environment of the Translation initiation site of the reporter gene (GUS) resulted in a fourfold increase in GUS expression in transformed rice cells. A combination of the optimized Translation initiation site and the Act1 intron resulted in a 40-fold increase in GUS expression by the CaMV35S promoter in transformed rice cells; similar Results were obtained using transformed maize cells. Overall, the investigations described above concluded that those based on the Actl promoter Expression vectors are suitable for a sufficiently strong and constitutive expression of foreign DNA in transformed cells to control monocotyledonous plants.

The expression cassette can advantageously one or more so-called "enhancer sequences" functionally linked to the Contain promoters that have an increased transgenic expression of the Enable nucleic acid sequence. Also at the 3 'end of the transgene expressing nucleic acid sequences can be additional advantageous sequences are inserted, such as further regulatory ones Elements or terminators. The transgene to be expressed Nucleic acid sequences can be in one or more copies in one of the Expression cassettes according to the invention may be included.

Control sequences are also to be understood as those which a homologous recombination or insertion into the genome of a Allow host organism or removal from the genome allow. In homologous recombination, for example, the natural promoter of a particular gene against one of the promoter according to the invention can be replaced. Methods like that cre / lox technology allow tissue-specific, under certain circumstances inducible removal of the expression cassette from the genome of the Host organism (Sauer B. (1998) Methods. 14 (4): 381-92). Here certain flanking sequences are added to the target gene (lox sequences), which are later removed using the Enable cre recombinase.

The promoter to be introduced can be homologous recombination are placed in front of the target gene to be expressed by: the promoter is linked to DNA sequences, for example are homologous to endogenous sequences that correspond to the reading frame of the Target gene are upstream. Such sequences are considered genetic Understand control sequences. After a cell with that the corresponding DNA construct has been transformed, the two can homologous sequences interact and thus the promoter sequence the desired location in front of the target gene so that the Promoter sequence now functionally linked to the target gene stands and forms an expression cassette according to the invention. The Selection of the homologous sequences determines the insertion site of the Promoter. Here, the expression cassette can be homologous Recombination using a simple or a double-reciprocal recombination are generated. With the simply reciprocal Recombination becomes just a single recombination sequence used and the entire inserted DNA is inserted. In the case of double-reciprocal recombination, the one to be introduced DNA flanked by two homologous sequences and the Insertion of the flanked area. The latter is procedure suitable to the natural promoter of a certain gene against one of the promoters according to the invention exchange and so the expression location and time of this gene modify. The functional link provides one Expression cassette according to the invention.

For the selection of successfully homologously recombined or transformed cells, it is usually necessary to use one selectable marker to introduce the successful recombined cells resistance to a biocide (for Example a herbicide), a metabolism inhibitor such as 2-Deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The Selection marker allows the selection of the transformed cells from untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84).

Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations in the host genome predominate. A chance that happens remove integrated sequences and thus cell clones with a Enriching correct homologous recombination consists in 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 one Recombination only between the two pairs of specific recombination sequences. The one pair of these specific ones DNA sequences will be outside the DNA sequence to be integrated, d. H. located outside of the two homologous DNA sequences. In the case of a correct homologous recombination, these become Sequences not transferred into the genome. In case of a random integration they usually insert together with the Rest of the construct. Using a special recombinase and a construct containing a second pair of specific sequences can be the randomly inserted sequences be cut out or inactivated by inversion while the sequences inserted correctly via homologous recombination in the Genome remain. A variety of sequence specific Recombination systems can be used, for example Bacteriophage P1 Cre / lox system, yeast FLP / FRT system, the Mu Phage gin recombinase, the E. pin recombinase called coli and the R / RS system of the pSR1 plasmid. Prefers are the bacteriophage P1 Cre / lox and the yeast FLP / FRT system. Here the recombinase (Cre or FLP) interacts specifically their respective recombination sequences (34bp lox sequence or 47 p FRT sequence) to the intermediate sequences delete or invert. The FLP / FRT and cre / lox Recombinase system has already been used in plant systems (Odell et al., Mol. Gen. Genet., 223: 369-378, 1990.)

Suitable polyadenylation signals as control sequences vegetable polyadenylation signals, preferably those which are in the essential T-DNA polyadenylation signals from Agrobacterium tumefaciens, especially gene 3 of 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 are the OCS (Octopine synthase) terminator and the NOS (Nopaline synthase) terminator.

• - Erleichtere Herstellung eines transgenen Organismus beispielsweise durch die Expression von Selektionsmarkern
• - Erzielen einer Resistenz gegen abiotische Stressfaktoren (Hitze, Kälte, Trockenheit, erhöhte Feuchtigkeit, Umweltgifte, UV-Strahlung)
• - Erzielen einer Resistenz gegen biotische Stressfaktoren (Pathogene, Viren, Insekten und Krankheiten)
• - Verbesserung von Nahrungs- oder Futtereigenschaften
• - Verbesserung der Wachstumsrate oder des Ertrages.

A large number of nucleic acids or proteins are known to the person skilled in the art, the recombinant expression of which, controlled by the expression cassettes or methods of the invention, is advantageous. Furthermore, a large number of genes are known to the person skilled in the art, by means of their repression or elimination by means of expression of a corresponding antisense RNA, advantageous effects can also be achieved. Examples include, but are not limited to, advantageous effects:

• - Easier production of a transgenic organism, for example, by the expression of selection markers
• - Achieve resistance to abiotic stress factors (heat, cold, dryness, increased moisture, environmental toxins, UV radiation)
• - Achieve resistance to biotic stress factors (pathogens, viruses, insects and diseases)
• - Improvement of food or feed properties
• - Improve growth rate or earnings.

Below are some concrete examples of nucleic acids called whose expression the desired beneficial effects provides:

1. Selection marker

Selection marker includes both positive selection markers, which are resistant to an antibiotic, herbicide or Give biocide, as well as negative selection markers, the one Giving sensitivity to these, as well as markers a growth advantage for the transformed organism grant (for example by expressing key genes of the cytokine; Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94: 2117-2121). Thrive in positive selection only the organisms that have the corresponding selection marker express, while in the negative selection it is precisely this received. In the production of transgenic plants Use of a positive selection marker preferred. It is also preferred to use selection markers which Give growth advantages. Negative selection markers can be used to advantage when it comes to determined Remove genes or sections of genomes from an organism (for example as part of an intersection process).

The selectable inserted with the expression cassette Marker gives the successfully recombined or transformed cells resistance to a biocide (for example a herbicide such as phosphinothricin, glyphosate or Bromoxynil), a metabolism inhibitor like 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic, for example Kanamycin, G 418, bleomycin, hygromycin. The selection marker allows the selection of the transformed cells from untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84). Particularly preferred selection markers are those confer resistance to herbicides. Exemplary as Selection markers are mentioned:

Numerous selection markers of this type are known to the person skilled in the art the known for these coding sequences. To be successors to name but not restrictive:

i) Positive selection markers

• - DNA Sequenzen, die für Phosphinothricinacetyltransferasen (PAT) kodieren, welche die freie Aminogruppe des Glutaminsynthaseinhibitors Phosphinothricin (PPT) acetyliert und damit eine Detoxifizierung des PPT erreicht (de Block et al. 1987, EMBO J. 6, 2513-2518) (auch Bialophos®resistenzgen (bar) genannt). Das bar Gen kodierend für eine Phosphinothricinacetyltransferase (PAT) kann aus beispielsweise Streptomyces hygroscopicus oder S. viridochromogenes isoliert werden. Entsprechende Sequenzen sind dem Fachmann bekannt (aus Streptomyces hygroscopicus Gen- Bank Acc.-No.: X17220 und X05822, aus Streptomyces viridochromogenes GenBank Acc.-No.: M 22827 und X65195; US 5,489,520). Ferner sind synthetische Gene beispielsweise für die Expression in Plastiden beschrieben AJ028212. Ein synthetisches Pat Gen ist beschrieben bei Becker et al. (1994), The Plant J. 5: 299-307. Ganz besonders bevorzugt ist ebenfalls die Expression des Polypeptides gemäß SEQ ID NO: 5, beispielsweise kodiert durch eine Nukleinsäuresequenz gemäß SEQ ID NO: 4. Die Gene verleihen Resistanz gegen das Herbizid Bialaphos oder Glufosinat und sind ein vielbenutzter Marker in transgenen Pflanzen (Vickers, JE et al. (1996). Plant Mol. Miol. reporter 14: 363-368; Thompson CJ et al. (1987) EMBO Journal 6: 2519-2523).
• - 5-Enolpyruvylshikimat-3-phosphatsynthasegene (EPSP Synthasegene), die eine Resistenz gegen Glyphosat® (N-(phosphonomethyl)glycin) verleihen. Das unselektive Herbizid Glyphosat hat 5-Enolpyruvyl-3-phosphoshikimatsynthase (EPSPS) als molekulares Target. Diese hat eine Schlüsselfunktion in der Biosynthese aromatischer Aminosäuren in Microben und Pflanzen, jedoch nicht in Säugern (Steinrucken HC et al. (1980) Biochem. Biophys. Res. Commun. 94: 1207-1212; Levin JG und. Sprinson DB (1964) J. Biol. Chem. 239: 1142-1150; Cole DJ (1985) Mode of action of glyphosate a literature analysis, p. 48-74. In: Grossbard E und Atkinson D (eds.). The herbicide glyphosate. Buttersworths, Boston.). Glyphosat-tolerante EPSPS Varianten werden bevorzugt als Selektionsmarker verwendet (Padgette SR et al. (1996). New weed control opportunities: development of soybeans with a Roundup Ready™ gene. In: Herbicide Resistant Crops (Duke, S.O., ed.), pp. 53-84. CRC Press, Boca Raton, FL; Saroha MK und Malik VS (1998) J Plant Biochemistry and Biotechnology 7: 65-72). Das EPSPS Gen des Agrobacterium sp. strain CP4 hat eine natürliche Toleranz gegen Glyphosat, die auf entsprechende transgene Pflanzen transferiert werden kann. Das CP4 EPSPS Gen wurde aus Agrobacterium sp. strain CP4 kloniert (Padgette SR et al. (1995)Crop Science 35(5): 1451-1461). 5-Enolpyrvylshikimate-3-phosphate-synthasen die Glyphosat-tolerant sind wie beispielsweise beschrieben in US 5,510,471; US 5,776,760; US 5,864,425; US 5,633,435; US 5,627; 061; US 5,463,175; EP 0 218 571, wobei die jeweils in den Patenten beschriebenen Sequenzen auch in der GenBank hinterlegt sind. Weitere Sequenzen sind beschrieben unter GenBank Accession X63374. Ferner ist das aroA Gen bevorzugt (M10947 S. typhimurium aroA locus 5-enolpyruvylshikimate-3-phosphate synthase (aroA protein) gene).
• - das für das Glyphosat® degradierende Enzyme kodierende gox Gen (Glyphosatoxidoreduktase). GOX (beispielsweise die Glyphosatoxidoreductase aus Achromobacter sp.) katalysiert die Spaltung einer C-N Bindung in Glyphosat was so zu Aminomethylphosphonsäure (AMPA) und Glyoxylat umgesetzt wird. GOX kann dadurch eine Resistenz gegen Glyphosat vermitteln (Padgette SR et al. (1996) J Nutr. 1996 Mar; 126(3): 702-16; Shah D et al. (1986) Science 233: 478-481).
• - das deh Gen (kodierend für eine Dehalogenase, die Dalapon® inaktiviert), (GenBank Acc.-No.: AX022822, AX022820 sowie WO99/27116)
• - bxn Gene, die für Bromoxynil® degradierende Nitrilaseenzyme kodieren. Beispielsweise die Nitrilase aus Klebstella ozanenae. Sequenzen sind in der genbank beispielsweise unter den Acc.-No: E01313 (DNA encoding promoxynil specific nitrilase) und J03196 (K.pneumoniae bromoxynilspecific nitrilase (bxn) gene, complete cds) zu finden.
• - Neomycinphosphotransferasen verleihen eine Resistenz gegen Antibiotika (Aminoglykoside) wie Neomycin, G418, Hygromycin, Paromomycin oder Kanamycin, indem sie durch eine Phosphorylierungsreaktion deren inhibierende Wirkung reduziert. Besonders bevorzugt ist das nptII Gen. Sequenzen können aus der GenBank erhalten werden (AF080390 Minitransposon mTn5-GNm; AF080389 Minitransposon mTn5-Nm, complete sequence). Zudem ist das Gen bereits Bestandteil zahlreicher Expressionsvektoren und kann unter Verwendung von dem Fachmann geläufigen Verfahren (wie beispielsweise Polymerasekettenreaktion) aus diesen isoliert werden (AF234316 pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). Das NPTII Gen kodiert für eine Aminoglycosid-3'O-phosphotransferase aus E.coli, Tn5 (GenBank Acc.- No: U00004 Position 1401-2300; Beck et al. (1982) Gene 19 327-336).
• - das DOG^R1-Gen. Das Gen DOG^R1 wurde aus der Hefe Saccharomyces cerevisiae isoliert (EP 0 807 836). Es codiert für eine 2-Desoxyglukose-6-phosphat Phosphatase, die Resistenz gegenüber 2-DOG verleiht (Randez-Gil et al. 1995, Yeast 11, 1233-1240; Sanz et al. (1994) Yeast 10: 1195-1202, Sequenz: GenBank Acc.-No.: NC001140 chromosom VIII, Saccharomyces cervisiae Position 194799-194056).
• - Sulfonylharnstoff- und Imidazolinon inaktivierende Acetolactatsynthasen, die eine Resistenz gegen Imidazolinon/Sulfonylharnstoff-Herbizide verleihen. Beispielhaft seien für Imidazolinon-Herbizide die Wirkstoffe Imazamethabenzmethyl, Imazzamox, Imazapyr, Imazaguin, Imazethapyr zu nennen. Für Sulfonylharnstoff-Herbizide seien beispielhaft Amidosulforon, Azimsulfuron, Chlorimuronethyl, Chlorsulfuron, Cinosulfuron, Imazosulforon, Oxasulforon, Prosulforon, Rimsulforon, Sulfosulforon zu nennen. Dem Fachmann sind zahlreiche weitere Wirkstoffe der genannten Klassen bekannt. Geeignet sind beispielsweise die Nukleinsäuren unter der GenBank Acc-No.: X51514 abgelegte Sequenz für das Arabidopsis thaliana Csr 1.2 Gen (EC 4.1.3.18) (Sathasivan K et al. (1990) Nucleic Acids Res. 18(8):2188). Acetolactatesynthasen die eine Resistenz gegen Imidazolinon-Herbizide verleihen sind ferner beschrieben unter den GenBank Acc.-No.:
  • a) AB049823 Oryza sativa ALS mRNA for acetolactate synthase, complete cds, herbicide resistant biotype
  • b) AF094326 Bassia scoparia herbicide resistant acetolactate synthase precursor (ALS) gene, complete cds
  • c) X07645 Tobacco acetolactate synthase gene, ALS SuRB (EC 4.1.3.18)
  • d) X07644 Tobacco acetolactate synthase gene, ALS SuRA (EC 4.1.3.18)
  • e) A19547 Synthetic nucleotide mutant acetolactate synthase
  • f) A19546 Synthetic nucleotide mutant acetolactate synthase
  • g) A19545 Synthetic nucleotide mutant acetolactate synthase
  • h) I05376 Sequence 5 from Patent EP 0257993
  • i) I05373 Sequence 2 from Patent EP 0257993
  • j) AL133315
  Bevorzugt ist die Expression einer Acetolactatsynthase gemäß SEQ ID NO: 7, beispielsweise kodiert durch eine Nukleinsäuresequenz gemäß SEQ ID NO: 6.
• - Hygromycinphosphotransferasen (X74325 P.pseudomallei gene for hygromycin phosphotransferase) die eine Resistenz gegen das Antibiotikum Hygromycin verleihen. Das Gen ist Bestandteil zahlreicher Expressionsvektoren und kann unter Verwendung von dem Fachmann geläufigen Verfahren (wie beispielsweise Polymerasekettenreaktion) aus diesen isoliert werden (AF294981 pINDEX4; AF234301 pCAMBIA-1380; AF234300 pCAMBIA-1304; AF234299 pCAMBIA-1303; AF234298 pCAMBIA-1302; AF354046 pCAMBIA-1305.; AF354045 pCAM- BIA-1305.1)
• - Resistenzgene gegen
  • 1. Chloramphenicol (Chloramphenicolacetyltransferase),
  • 2. Tetracyclin, verschiedene Resistenzgene sind beschrieben z. B. X65876 S. ordonez genes class D tetA and tetR for tetracycline resistance and repressor proteins X51366 Bacillus cereus plasmid pBC16 tetracycline resistance gene. Zudem ist das Gen bereits Bestandteil zahlreicher Expressionsvektoren und kann unter Verwendung von dem Fachmann geläufigen Verfahren (wie beispielsweise Polymerasekettenreaktion) aus diesen isoliert werden
  • 3. Streptomycin, verschiedene Resistenzgene sind beschrieben z. B. mit der GenBank Acc.-No.: AJ278607 Corynebacterium acetoacidophilum ant gene for streptomycin adenylyltransferase.
  • 4. Zeocin, das entsprecende Resistenzgen ist Bestandteil zahlreicher Klonierungsvektoren (z. B. L36849 Cloning vector pZEO) und kann unter Verwendung von dem Fachmann geläufigen Verfahren (wie beispielsweise Polymerasekettenreaktion) aus diesen isoliert werden.
  • 5. Ampicillin (β-Lactamase Gen; Datta N, Richmond HR. (1966) Biochem J. 98(1): 204-9; Heffron F et al (1975) J. Bacteriol 122: 250-256; das Amp Gen wurde zuerst zur Herstellung des E. coli Vektors pBR322 kloniert; Bolivar F et al. (1977) Gene 2: 95-114). Die Sequenz ist Bestandteil zahlreicher Klonierungsvektoren und kann unter Verwendung von dem Fachmann geläufigen Verfahren (wie beispielsweise Polymerasekettenreaktion) aus diesen isoliert werden.
• - Gene wie die Isopentenyltransferase aus Agrobacterium tumefaciens (strain: PO22) (Genbank Acc.-No.: AB025109). Das ipt Gen ist ein Schlüsselenzym der Cytokin-Biosynthese. Seine Überexpression erleichtert die Regeneration von Pflanzen (z. B. Selektion auf Cytokin-freien Medium). Das Verfahren zur Nutzung des ipt Gens ist beschrieben (Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94: 2117-2121; Ebinuma, H et al. (2000) Selection of Marker-free transgenic plants using the oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable markers, In Molecular Biology of Woody Plants. Kluwer Academic Publishers).

The selectable marker introduced with the expression cassette gives the successfully recombined or transformed cells resistance to a biocide (for example a herbicide such as phosphinothricin, glyphosate or bromoxynil), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or Antibiotic, such as tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin or hygromycin. The selection marker allows the selection of the transformed cells from untransformed ones (McCormick et al., Plant Cell Reports 5 (1986), 81-84). Particularly preferred selection markers are those which confer resistance to herbicides. Examples of selection markers are:

• - DNA sequences which code for phosphinothricin acetyltransferases (PAT), which acetylates the free amino group of the glutamine synthase inhibitor phosphinothricin (PPT) and thus detoxifies the PPT (de Block et al. 1987, EMBO J. 6, 2513-2518) (also Bialophos ®resistance gene (bar) called). The bar gene coding for a phosphinothricin acetyltransferase (PAT) can be isolated from, for example, Streptomyces hygroscopicus or S. viridochromogenes. Corresponding sequences are known to the person skilled in the art (from Streptomyces hygroscopicus GenBank Acc.-No .: X17220 and X05822, from Streptomyces viridochromogenes GenBank Acc.-No .: M 22827 and X65195; US 5,489,520). Furthermore, synthetic genes are described, for example, for expression in plastids AJ028212. A synthetic Pat gene is described by Becker et al. (1994), The Plant J. 5: 299-307. Expression of the polypeptide according to SEQ ID NO: 5 is also very particularly preferred, for example encoded by a nucleic acid sequence according to SEQ ID NO: 4. The genes confer resistance to the herbicide bialaphos or glufosinate and are a widely used marker in transgenic plants (Vickers, JE et al. (1996) Plant Mol. Miol. reporter 14: 363-368; Thompson CJ et al. (1987) EMBO Journal 6: 2519-2523).
• - 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which confer resistance to Glyphosat® (N- (phosphonomethyl) glycine). The unselective herbicide glyphosate has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) as its molecular target. This has a key function in the biosynthesis of aromatic amino acids in microbes and plants, but not in mammals (Steinrucken HC et al. (1980) Biochem. Biophys. Res. Commun. 94: 1207-1212; Levin JG and. Sprinson DB (1964) J. Biol. Chem. 239: 1142-1150; Cole DJ (1985) Mode of action of glyphosate a literature analysis, p. 48-74. In: Grossbard E and Atkinson D (eds.). The herbicide glyphosate. Buttersworths, Boston.). Glyphosate-tolerant EPSPS variants are preferably used as selection markers (Padgette SR et al. (1996). New weed control opportunities: development of soybeans with a Roundup Ready ™ gene. In: Herbicide Resistant Crops (Duke, SO, ed.), Pp 53-84 CRC Press, Boca Raton, FL; Saroha MK and Malik VS (1998) J Plant Biochemistry and Biotechnology 7: 65-72). The EPSPS gene of Agrobacterium sp. strain CP4 has a natural tolerance to glyphosate, which can be transferred to corresponding transgenic plants. The CP4 EPSPS gene was derived from Agrobacterium sp. strain CP4 cloned (Padgette SR et al. (1995) Crop Science 35 (5): 1451-1461). 5-enolpyrvylshikimate-3-phosphate synthases which are glyphosate-tolerant as described, for example, in US 5,510,471; US 5,776,760; US 5,864,425; US 5,633,435; US 5,627; 061; US 5,463,175; EP 0 218 571, the sequences described in the patents also being stored in the GenBank. Further sequences are described under GenBank Accession X63374. The aroA gene is also preferred (M10947 S. typhimurium aroA locus 5-enolpyruvylshikimate-3-phosphate synthase (aroA protein) gene).
• - the gox gene (glyphosate oxidoreductase) coding for the glyphosate® degrading enzyme. GOX (for example the glyphosate oxidoreductase from Achromobacter sp.) Catalyzes the cleavage of a CN bond into glyphosate, which is converted into aminomethylphosphonic acid (AMPA) and glyoxylate. GOX can thereby confer resistance to glyphosate (Padgette SR et al. (1996) J Nutr. 1996 Mar; 126 (3): 702-16; Shah D et al. (1986) Science 233: 478-481).
• - the deh gene (coding for a dehalogenase which inactivates Dalapon®), (GenBank Acc.-No .: AX022822, AX022820 and WO99 / 27116)
• - bxn genes which code for Bromoxynil® degrading nitrilase enzymes. For example, the nitrilase from Klebstella ozanenae. Sequences can be found in the genebank, for example, under Acc.-No: E01313 (DNA encoding promoxynil specific nitrilase) and J03196 (K.pneumoniae bromoxynilspecific nitrilase (bxn) gene, complete cds).
• - Neomycin phosphotransferases confer resistance to antibiotics (aminoglycosides) such as neomycin, G418, hygromycin, paromomycin or kanamycin by reducing their inhibitory effect through a phosphorylation reaction. The nptII gene is particularly preferred. Sequences can be obtained from GenBank (AF080390 mini transposon mTn5-GNm; AF080389 mini transposon mTn5-Nm, complete sequence). In addition, the gene is already part of numerous expression vectors and can be isolated from them using methods familiar to the person skilled in the art (such as, for example, polymerase chain reaction) (AF234316 pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). The NPTII gene codes for an aminoglycoside-3'O-phosphotransferase from E. coli, Tn5 (GenBank Acc.-No: U00004 position 1401-2300; Beck et al. (1982) Gene 19 327-336).
• - the DOG ^R 1 gene. The gene DOG ^R 1 was isolated from the yeast Saccharomyces cerevisiae (EP 0 807 836). It encodes a 2-deoxyglucose-6-phosphate phosphatase that confers resistance to 2-DOG (Randez-Gil et al. 1995, Yeast 11, 1233-1240; Sanz et al. (1994) Yeast 10: 1195-1202, Sequence: GenBank Acc.-No .: NC001140 chromosome VIII, Saccharomyces cervisiae position 194799-194056).
• - Sulfonylurea and imidazolinone inactivating acetolactate synthases which confer resistance to imidazolinone / sulfonylurea herbicides. The active substances imazamethabenzmethyl, imazzamox, imazapyr, imazaguin and imazethapyr are examples of imidazolinone herbicides. Examples of sulfonylurea herbicides are amidosulforon, azimsulfuron, chlorimuronethyl, chlorosulfuron, cinosulfuron, imazosulforon, oxasulforon, prosulforon, rimsulforon, sulfosulforon. Numerous other active substances of the classes mentioned are known to the person skilled in the art. For example, the nucleic acids under GenBank Acc-No .: X51514 are suitable sequences for the Arabidopsis thaliana Csr 1.2 gene (EC 4.1.3.18) (Sathasivan K et al. (1990) Nucleic Acids Res. 18 (8): 2188). Acetolactate synthases which confer resistance to imidazolinone herbicides are also described under GenBank Acc.-No .:
  • a) AB049823 Oryza sativa ALS mRNA for acetolactate synthase, complete cds, herbicide resistant biotype
  • b) AF094326 Bassia scoparia herbicide resistant acetolactate synthase precursor (ALS) gene, complete cds
  • c) X07645 Tobacco acetolactate synthase gene, ALS SuRB (EC 4.1.3.18)
  • d) X07644 Tobacco acetolactate synthase gene, ALS SuRA (EC 4.1.3.18)
  • e) A19547 synthetic nucleotide mutant acetolactate synthase
  • f) A19546 synthetic nucleotide mutant acetolactate synthase
  • g) A19545 synthetic nucleotide mutant acetolactate synthase
  • h) I05376 Sequence 5 from Patent EP 0257993
  • i) I05373 Sequence 2 from Patent EP 0257993
  • j) AL133315
  Expression of an acetolactate synthase according to SEQ ID NO: 7 is preferred, for example encoded by a nucleic acid sequence according to SEQ ID NO: 6.
• - Hygromycin phosphotransferases (X74325 P. pseudomallei gene for hygromycin phosphotransferase) which confer resistance to the antibiotic hygromycin. The gene is part of numerous expression vectors and can be isolated from them using methods known to the person skilled in the art (such as, for example, polymerase chain reaction) (AF294981 pINDEX4; AF234301 pCAMBIA-1380; AF234300 pCAMBIA-1304; AF234299 pCAMBIA-1303; AF234298 pCAMBIA-130) -1305 .; AF354045 pCAM- BIA-1305.1)
• - resistance genes against
  • 1. chloramphenicol (chloramphenicol acetyl transferase),
  • 2. Tetracycline, various resistance genes are described for. B. X65876 S. ordonez genes class D tetA and tetR for tetracycline resistance and repressor proteins X51366 Bacillus cereus plasmid pBC16 tetracycline resistance gene. In addition, the gene is already part of numerous expression vectors and can be isolated from these using methods familiar to the person skilled in the art (such as, for example, polymerase chain reaction)
  • 3. Streptomycin, various resistance genes are described e.g. B. with GenBank Acc.-No .: AJ278607 Corynebacterium acetoacidophilum ant gene for streptomycin adenylyl transferase.
  • 4. Zeocin, the corresponding resistance gene is a component of numerous cloning vectors (for example L36849 cloning vector pZEO) and can be isolated from these using methods which are familiar to the person skilled in the art (such as, for example, polymerase chain reaction).
  • 5. Ampicillin (β-lactamase gene; Datta N, Richmond HR. (1966) Biochem J. 98 (1): 204-9; Heffron F et al (1975) J. Bacteriol 122: 250-256; the Amp gene was first cloned to produce the E. coli vector pBR322; Bolivar F et al. (1977) Gene 2: 95-114). The sequence is part of numerous cloning vectors and can be isolated from them using methods familiar to those skilled in the art (such as, for example, polymerase chain reaction).
• - Genes such as the isopentenyl transferase from Agrobacterium tumefaciens (strain: PO22) (Genbank Acc.-No .: AB025109). The ipt gene is a key enzyme in cytokine biosynthesis. Its overexpression facilitates the regeneration of plants (e.g. selection on cytokine-free medium). The procedure for using the ipt gene is described (Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94: 2117-2121; Ebinuma, H et al. (2000) Selection of Marker-free transgenic plants using the oncogenes (ipt , rol A, B, C) of Agrobacterium as selectable markers, In Molecular Biology of Woody Plants. Kluwer Academic Publishers).

ii) Negative selection markers

Negative selection markers allow, for example, the Selection of organisms with successfully deleted sequences, comprising the marker gene (Koprek T et al. (1999) The Plant Journal 19 (6) 719-726). TK thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), coding for codA gene for a cytosine deaminase (Gleave AP et al. (1999) Plant Mol Biol. 40 (2): 223-35; Perera RJ et al. (1993) Plant Mol. Biol 23 (4): 793-799; Stougaard J; (1993) Plant J 3: 755-761) that Cytochrome P450 gene (Koprek et al. (1999) Plant J. 156: 719-726), genes coding for a haloalkane dehalogenase (Naested H (1999) Plant J. 18: 571-576), the iaaH gene (Sundaresan V et al. (1995) Genes & Development 9: 1797-1810) or the tms2 gene (Fedoroff NV & Smith DL 1993, Plant J 3: 273-289).

The concentrations of the used for the selection Antibiotics, herbicides, biocides or toxins must be added to the be adapted to the respective test conditions or organisms. Kanamycin (Km) 50 should be mentioned as an example for plants mg / l, hygromycin B 40 mg / l, phosphinothricin (Ppt) 6 mg / l.

• 1. Verbesserter Schutz der Pflanze gegen abiotische Stressfaktoren wie Trockenheit, Hitze, oder Kälte zum Beispiel durch Überexpression von dem "antifreeze polypeptides aus Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, dem Arabidopsis thaliana Transkriptionsaktivator CBF1, Glutamatdehydrogenasen (WO 97/12983, WO 98/11240), Calciumabhängigen Proteinkinasegenen (WO 98/26045), Calcineurinen (WO 99/05902), Farnesyltransferasen (WO 99/06580), Pei ZM et al., Science 1998, 282: 287-290), Ferritin (Deak M et al., Nature Biotechnology 1999, 17: 192-196), Oxalatoxidase (WO 99/04013; Dunwell JM Biotechnology and Genetic Engeneering Reviews 1998, 15: 1-32), DREB1A-Faktor (dehydration response element B 1A; Kasuga M et al., Nature Biotechnology 1999, 17: 276-286), Genen der Mannitol- oder Trehalosesynthese wie der Trehalosephosphatsynthase oder der Trehalosephosphatphosphatase (WO 97/42326), oder durch Inhibition von Genen wie der Trehalase (WO 97/50561). Besonders bevorzugt sind Nukleinsäuren, die für den transkriptionellen Aktivator CBF1 aus Arabidopsis thaliana (GenBank Acc.-No.: U77378) oder das "antifreeze protein" aus Myoxocephalus octodecemspinosus (GenBank Acc.-No.: AF306348) oder funktionelle Äquivalente derselben kodieren.
• 2. Expression von Stoffwechselenzymen zur Verwendung im Futter- und Nahrungsmittelbereich, zum Beispiel die Expression von Phytase und Cellulasen. Besonders bevorzugt sind Nukleinsäuren wie die künstliche für eine mikrobielle Phytase kodierende cDNA (GenBank Acc.-No.: A19451) oder funktionelle Äquivalente derselben.
• 3. Erreichen einer Resistenz zum Beispiel gegen Pilze, Insekten, Nematoden und Krankheiten durch gezielte Absonderung oder Anreicherung bestimmter Metaboliten oder Proteine in der Epidermis des Embryos. Beispielhaft seien genannt Glucosinolate (Abwehr von Herbivoren), Chitinasen oder Glucanasen und andere Enzyme die die Zellwand von Parasiten zerstören, Ribosom-inaktivierende Proteine (RIPs) und andere Proteine der pflanzlichen Resistenz- und Stressreaktion, wie sie bei Verletzung oder mikrobiellen Befall von Pflanzen oder chemisch durch zum Beispiel Salicylsäure, Jasmonsäure oder Ethylen induziert werden, Lysozyme aus nicht-pflanzlichen Quellen wie zum Beispiel T4 Lysozym oder Lysozm aus verschiedenenen Säugern, insektizide Proteine wie Bacillus thuringiensis Endotoxin, α-Amylaseinhibitor oder Proteaseinhibitoren (cowpea Trypsininhibitor), Glucanasen, Lectine wie Phytohemagglutinin, Schneeglöckchenlectin, Weizenkeimagglutinin, RNAsen oder Ribozyme. Besonders bevorzugt sind Nukleinsäuren, die für die chit42 Endochitinase aus Trichoderma harzianum (GenBank Acc.- No.: 578423) oder für das N-hydroxylierende, multifunktionelle Cytochrom P-450 (CYP79) Proteine aus Sorghum bicolor (GenBank Acc.-No.: U32624) oder funktionelle Äquivalente derselben kodieren.
  Bekannt ist die Akkumulation von Glucosinolaten in Pflanzen der Gattung der Cardales insbesondere der Ölsaaten zum Schutz vor Schädlingen (Rask L et al. (2000) Plant Mol Biol 42: 93-113; Menard R et al. (1999) Phytochemistry 52: 29-35), die Expression des Bacillus thuringiensis Endotoxin unter Kontrolle des 35 S CaMV Promotors (Vaeck et al. (1987) Nature 328: 33-37) oder der Schutz des Tabaks gegen Pilzbefall durch Expression einer Chitinase aus der Bohne unter Kontrolle des CaMV Promotors (Broglie et al. (1991) Science 254: 1194-1197).
  Durch Expression des Lectins Agglutinin aus Schneeglöckchen (Galanthus nivalis) kann eine Resistenz gegen Schädlinge wie den Reisschädling Nilaparvata lugens beispielsweise in transgenen Reispflanzen erreicht werden (Rao et al. (1998) Plant J. 15(4): 469-77.). Nilaparvata lugens zählt zu den Phloem- saugenden Schädlingen und fungiert darüberhinaus als Überträger bedeutender Virus-bedingter Pflanzenkrankheiten.
  Die Expression synthetischer cryIA(b) and cryIA(c) Gene, die für Lepidopteren-spezifische delta-Endotoxine aus Bacillus thuringiensis kodieren, kann in verschiedene Pflanzen eine Resistenz gegen Schadinsekten bewirken. So kann in Reis eine Resistenz gegen zwei der wichtigsten Reisschädlinge, den gestreiften Stengelbohrer (Chilo suppressalis) und den gelben Stengelbohrer (Scirpophaga incertulas), erzielt werden (Cheng X et al. (1998) Proc Natl Acad Sci USA 95(6): 2767-2772; Nayak P et al. (1997) Proc Natl Acad Sci USA 94(6): 2111-2116).
• 4. Expression von Genen, die eine Akkumulation von Feinchemikalien, wie von Tocopherolen, Tocotrienolen oder Carotiniden bewirken. Beispielhaft sei die Phytoendesaturase genannt. Bevorzugt sind Nukleinsäuren, die für die Phytoendesaturase aus Narcissus pseudonarcissus (GenBank Acc.-No.: X78815) oder funktionelle Äquivalente derselben kodieren.
• 5. Produktion von Neutraceuticals wie zum Beispiel polyungesättigten Fettsäuren wie beispielsweise Arachidonsäure oder EP (Eicosapentaensäure) oder DHA (Docosahexaensäure) durch Expression von Fettsäureelongasen und/oder -desaturasen oder Produktion von Proteinen mit verbessertem Nahrungswert wie zum Beispiel mit einem hohen Anteil an essentiellen Aminosäuren (z. B. das methioninreiche 2S Albumingens der Brasilnuss). Bevorzugt sind Nukleinsäuren, die für das methioninreiche 2S Albumin aus Bertholletia excelsa (GenBank Acc.-No.: AB044391), die Δ6-Acyllipiddesaturase aus Physcomitrella patens (GenBank Acc.-No.: AJ222980; Girke et al 1998, The Plant Journal 15: 39-48), die Δ6-Desaturase aus Mortierella alpina (Sakuradani et al 1999 Gene 238: 445-453), die Δ5-Desaturase aus Caenorhabditis elegans (Michaelson et al. 1998, FEBS Letters 439: 215-218), die Δ5-Fettsäuredesaturase (des-5) aus Caenorhabditis elegans (GenBank Acc.-No.: AF078796), die Δ5-Desaturase aus Mortierella alpina (Michaelson et al. JBC 273: 19055-19059), die Δ6-Elongase aus Caenorhabditis elegans (Beaudoin et al. 2000, PNAS 97: 6421-6426), die Δ6-Elongase aus Physcomitrella patens (Zank et al. 2000, Biochemical Society Transactions 28: 654-657) oder funktionelle Äquivalente derselben kodieren.
• 6. Produktion von Feinchemikalien (wie beispielsweise Enzymen) und Pharmazeutika (wie zum Beispiel Antikörpern oder Vakzinen wie beschrieben bei Hood EE, Jilka JM. (1999) Curr Opin Biotechnol. 10(4): 382-6; Ma JK, Vine ND (1999) Curr Top Microbiol Immunol 236: 275-92). Beispielsweise konnte rekombinantes Avidin aus Hühnereiweiß und bakterieller β-Glucuronidase (GUS) in großen Maßstab in transgenen Maispflanzen produziert werden (Hood et al. (1999) Adv Exp Med Biol 464: 127-47. Review.). Diese rekombinanten Proteine aus Maispflanzen werden von der Firma Sigma (Sigma Chemicals Co.) als hochreine Biochemikalien vertrieben.
• 7. Erreichen einer erhöhten Speicherfähigkeit in Zellen, die normalerweise weniger Speicherproteine oder -lipide enthalten mit dem Ziel, den Ertrag an diesen Substanzen zu erhöhen, zum Beispiel durch Expression eine Acetyl-CoA Carboxylase. Bevorzugt sind Nukleinsäuren, die für die Acetyl-CoA Carboxylase (Accase) aus Medicago sativa (GenBank Acc.-No.: L25042) oder funktionelle Äquivalente derselben kodieren.

Furthermore, functional analogs of the nucleic acids mentioned can be expressed coding for selection markers. Functional analogs here means all the sequences which have essentially the same function, ie are capable of selecting transformed organisms. The functional analogue can differ in other characteristics. For example, it may have a higher or lower activity, or it may have other functionalities.

• 1. Improved protection of the plant 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 transcription activator CBF1, glutamate dehydrogenases (WO 97/00512) , WO 98/11240), calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), farnesyltransferases (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 Biotechnology and Genetic Engeneering Reviews 1998, 15: 1-32), DREB1A factor (dehydration response element B 1A; Kasuga M et al., Nature Biotechnology 1999, 17: 276-286), genes of mannitol 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) Especially before There are nucleic acids encoding the transcriptional activator CBF1 from Arabidopsis thaliana (GenBank Acc.-No .: U77378) or the "antifreeze protein" from Myoxocephalus octodecemspinosus (GenBank Acc.-No .: AF306348) or functional equivalents thereof.
• 2. Expression of metabolic enzymes for use in the feed and food sector, for example the expression of phytase and cellulases. Nucleic acids such as the artificial cDNA coding for a microbial phytase (GenBank Acc.-No .: A19451) or functional equivalents thereof are particularly preferred.
• 3. Achieving resistance, for example, to fungi, insects, nematodes and diseases through the targeted secretion or accumulation of certain metabolites or proteins in the epidermis of the embryo. Examples include glucosinolates (defense against herbivores), chitinases or glucanases and other enzymes that destroy the cell wall of parasites, ribosome-inactivating proteins (RIPs) and other proteins of the plant resistance and stress reaction, such as those caused by injury or microbial attack on plants or chemically induced by, for example, salicylic acid, jasmonic acid or ethylene, lysozymes from non-plant sources such as, for example, T4 lysozyme or lysozyme from various mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, α-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor L, such as gline) Phytohemagglutinin, snowdrop lectin, wheat germ agglutinin, RNAsen or ribozymes. Nucleic acids which are particularly preferred are those for the chit42 endochitinase from Trichoderma harzianum (GenBank Acc.-No .: 578423) or for the N-hydroxylating, multifunctional cytochrome P-450 (CYP79) proteins from Sorghum bicolor (GenBank Acc.-No .: U32624) or encode their functional equivalents.
  The accumulation of glucosinolates in plants of the Cardales genus, in particular oilseeds for protection against pests, is known (Rask L et al. (2000) Plant Mol Biol 42: 93-113; Menard R et al. (1999) Phytochemistry 52: 29- 35), the expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al. (1987) Nature 328: 33-37) or the protection of the tobacco against fungal attack by expression of a chitinase from the bean under the control of the CaMV promoter (Broglie et al. (1991) Science 254: 1194-1197).
  Expression of the lectin agglutinin from snowdrops (Galanthus nivalis) can achieve resistance to pests such as the rice pest Nilaparvata lugens, for example, in transgenic rice plants (Rao et al. (1998) Plant J. 15 (4): 469-77.). Nilaparvata lugens is one of the phloem-sucking pests and also acts as a carrier of important virus-related plant diseases.
  The expression of synthetic cryIA (b) and cryIA (c) genes which code for lepidopteran-specific delta endotoxins from Bacillus thuringiensis can cause resistance to insect pests in various plants. In rice, resistance to two of the most important rice pests, the striped stem borer (Chilo suppressalis) and the yellow stem borer (Scirpophaga incertulas), can be achieved (Cheng X et al. (1998) Proc Natl Acad Sci USA 95 (6): 2767 -2772; Nayak P et al. (1997) Proc Natl Acad Sci USA 94 (6): 2111-2116).
• 4. Expression of genes which cause the accumulation of fine chemicals, such as tocopherols, tocotrienols or carotenoids. The phytoendesaturase may be mentioned as an example. Preferred are nucleic acids which code for the phytoendesaturase from Narcissus pseudonarcissus (GenBank Acc.-No .: X78815) or functional equivalents thereof.
• 5.Production of neutraceuticals such as polyunsaturated fatty acids such as arachidonic acid or EP (eicosapentaenoic acid) or DHA (docosahexaenoic acid) by expressing fatty acid elongases and / or desaturases or production of proteins with improved nutritional value such as a high proportion of essential amino acids ( e.g. the methionine-rich 2S albumingens of 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 Caenorhabditis 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), which encode Δ6 elongase from Physcomitrella patens (Zank et al. 2000, Biochemical Society Transactions 28: 654-657) or functional equivalents thereof.
• 6. Production of fine chemicals (such as enzymes) and pharmaceuticals (such as antibodies or vaccines as described in Hood EE, Jilka JM. (1999) Curr Opin Biotechnol. 10 (4): 382-6; Ma JK, Vine ND ( 1999) Curr Top Microbiol Immunol 236: 275-92). For example, recombinant avidin from chicken protein and bacterial β-glucuronidase (GUS) could be produced on a large scale in transgenic maize plants (Hood et al. (1999) Adv Exp Med Biol 464: 127-47. Review.). These recombinant proteins from maize plants are sold by Sigma (Sigma Chemicals Co.) as high-purity biochemicals.
• 7. Achieving increased storage capacity in cells that normally contain fewer 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.

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

Furthermore, functional analogs of the nucleic acids mentioned can or proteins are expressed. Functional analogs mean here all the sequences that have essentially the same function d. H. to the function (for example a substrate conversion or a signal transduction) are also capable as an example called protein. The functional analogue can distinguish other characteristics. For example it can have a higher or lower activity or also about have additional functionalities. Functional analogues also means Sequences for fusion proteins consisting of one of the preferred proteins and other proteins for example another preferred protein or a signal peptide sequence encode.

• a) kleine Untereinheit (SSU) der Ribulosebisphosphatcarboxylase (Rubisco ssu) aus Erbse, Mais, Sonnenblume
• b) Transitpeptide abgeleitet von Genen der pflanzlichen Fettsäurebiosynthese wie das Transitpeptid des plastidären "Acyl Carrier Protein" (ACP), die Stearyl-ACP-Desaturase, β-Ketoacyl-ACP Synthase oder die Acyl-ACP-Thioesterase.
• c) das Transitpeptid für GBSSI ("Starch Granule Bound Synthase I")
• d) LHCP II Gene.

The expression of the nucleic acids under the control of the promoters according to the invention is in any desired cell compartment, such as. B. the endomembrane system, the vacuole and the chloroplasts possible. By using the secretory path desired glycosylation reactions, special folds and. possible. Secretion of the target protein to the cell surface or secretion into the culture medium, for example when using suspension-cultured cells or protoplasts, is also possible. The target sequences required for this can be taken into account in individual vector variations as well as introduced into the vector together with the target gene to be cloned by using a suitable cloning strategy. Both target genes, if available, or heterologous sequences can be used as target sequences. Additional, heterologous, but not limited to, sequences that are preferred for functional linkage are further targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments ; and translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711) and the like. The method of specifically transporting proteins that are not per se localized in the plastids into the plastids is described (Klosgen RB and Weil JH (1991) Mol Gen Genet 225 (2): 297-304; Van Breusegem F et al. (1998) Plant Mol Biol. 38 (3): 491-496). Preferred sequences are:

• a) Small subunit (SSU) of ribulose bisphosphate carboxylase (Rubisco ssu) from pea, corn, sunflower
• b) Transit peptides derived from genes of plant fatty acid biosynthesis such as the transit peptide of the plastid "acyl carrier protein" (ACP), the stearyl-ACP desaturase, β-ketoacyl-ACP synthase or the acyl-ACP thioesterase.
• c) the transit peptide for GBSSI ("Starch Granule Bound Synthase I")
• d) LHCP II genes.

The target sequences can be shared with others from the transit peptide coding sequences linked to different targeting sequences to be a subcellular localization in the apoplast, in the Vacuole, in plastids, in the mitochondrion, in the endoplasmic Reticulum (ER), in the cell nucleus, in oil corpuscles or others To ensure compartments. Furthermore, as well Translation enhancers such as the 5 'leader sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711) and the like are used.

The person skilled in the art is also aware that he described the above Genes not directly using the coding for these genes Express nucleic acid sequences or for example by antisense must repress. It can also be artificial, for example Use zinc finger protein type transcription factors (Beerli RR et al. (2000) Proc Natl Acad Sci USA 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 an expression or Repression of the endogenous gene. So you can get the ones you want Effects also by expression of a corresponding zinc finger Transcription factor under the control of one of the invention Reach promoters.

The expression cassettes according to the invention can also be used for Suppression or reduction of replication or / and translation of target genes can be used by "gene silencing".

The expression cassettes according to the invention can also be used to express nucleic acids, the so-called Mediate "antisense" effects and thus, for example, for reduction are capable of expressing a target protein.

• a) Polygalakturonase zur Verhinderung von Zellabbau und "Matschig"-werden von Pflanzen und Früchten bespielsweise Tomaten. Bevorzugt werden dazu Nukleinsäuresequenzen wie die des Polygalakturonase-Gens der Tomate (GenBank Acc.-No.: X14074) oder dessen Homologe aus anderen Gattungen und Arten verwendet.
• b) Verminderung der Expression von allergenen Proteinen wie beispielsweise beschrieben bei Tada Y et al. (1996) FEBS Lett 391(3): 341-345 oder Nakamura R (1996) Biosci Biotechnol Biochem 60(8): 1215-1221ö.
• c) Veränderung der Blütenfarbe durch Suppression der Expression von Enzymen der Anthocyanbiosynthese. Entsprechende Vorgehensweisen sind beschrieben (beispielsweise bei Forkmann G, Martens S. (2001) Curr Opin Biotechnol 12(2): 155-160). Bevorzugt werden dazu Nukleinsäuresequenzen wie die der Flavonoid-3'-hydroxylase (GenBank Acc.-No.: AB045593), der Dihydroflavanol-4-reduktase (GenBank Acc.-No.: AF017451), der Chalconisomerase (GenBank Acc.-No.: AF276302), der Chalconsynthase (GenBank Acc.-No.: AB061022), der Flavanone-3-betahydroxylase (GenBank Acc.-No.: X72592) oder der Flavonesynthase II (GenBank Acc.-No.: AB045592) oder dessen Homologe aus anderen Gattungen und Arten verwendet.
• d) Verschiebung des Amylose/Amylopektingehaltes in Stärke durch Suppression des Verzweigungsenzyms Q, das für die α-1,6-glykosidische Verknüpfung verantwortlich ist. Entsprechende Vorgehensweisen sind beschrieben (beispielsweise bei Schwall GP et al. (2000) Nat Biotechnol 18(5): 551-554). Bevorzugt werden dazu Nukleinsäuresequenzen wie die des Starch branching enzyme II der Kartoffel (GenBank Acc.-No.: AR123356; US 6,169,226) oder dessen Homologe aus anderen Gattungen und Arten verwendet.

Preferred genes or proteins whose suppression requires an advantageous phenotype include, by way of example, but not by way of limitation:

• a) Polygalacturonase to prevent cell degradation and "mushy" plants and fruits, for example tomatoes. Nucleic acid sequences such as that of the tomato polygalacturonase gene (GenBank Acc. No .: X14074) or its homologs from other genera and species are preferably used for this purpose.
• b) Reduction of the expression of allergenic proteins as described for example in Tada Y et al. (1996) FEBS Lett 391 (3): 341-345 or Nakamura R (1996) Biosci Biotechnol Biochem 60 (8): 1215-1221ö.
• c) Change in flower color by suppression of the expression of enzymes in anthocyanbiosynthesis. Appropriate procedures are described (for example, in Forkmann G, Martens S. (2001) Curr Opin Biotechnol 12 (2): 155-160). Nucleic acid sequences such as that of flavonoid 3'-hydroxylase (GenBank Acc.-No .: AB045593), dihydroflavanol-4-reductase (GenBank Acc.-No .: AF017451), chalcone isomerase (GenBank Acc.-No. : AF276302), chalcone synthase (GenBank Acc.-No .: AB061022), flavanone-3-beta-hydroxylase (GenBank Acc.-No .: X72592) or Flavonesynthase II (GenBank Acc.-No .: AB045592) or its homologue used from other genera and species.
• d) Shift in the amylose / amylopectin content in starch by suppression of the branching enzyme Q, which is responsible for the α-1,6-glycosidic linkage. Appropriate procedures are described (for example in Schwall GP et al. (2000) Nat Biotechnol 18 (5): 551-554). Nucleic acid sequences such as that of the Starch branching enzyme II of the potato (GenBank Acc.-No .: AR123356; US 6,169,226) or its homologs from other genera and species are preferably used for this purpose.

An "antisense" nucleic acid means one at first Nucleic acid sequence wholly or partly to at least part of the "sense" strands of said target protein is complementary. the Those skilled in the art are known to use alternative cDNA or the corresponding gene as the starting template for corresponding antisense Can use constructs. The "antisense" is preferred Nucleic acid complementary to the coding region of the target protein or part of the same. The "antisense" nucleic acid can also to the non-coding region or a part thereof to be complementary. Based on the sequence information for a Target protein, can contain an antisense nucleic acid Consideration of the base pair rules of Watson and Crick in the expert be designed in a common manner. An antisense nucleic acid can be complementary to all or part of the Nucleic acid sequence of a target protein. In a preferred one In one embodiment, the antisense nucleic acid is an oligonucleotide a length of, for example, 15, 20, 25, 30, 35, 40, 45 or 50 Nucleotides.

The antisense nucleic acid comprises in a preferred Embodiment α-anomeric nucleic acid molecules. α-anomer Nucleic acid molecules form special double-stranded hybrids complementary RNA in which, in contrast to the normal β-units Strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-6641).

The use of those described above is also included Sequences in sense orientation, which is familiar to the person skilled in the art can lead to co-suppression. In tobacco, tomato and petunia is it has been demonstrated that the expression of sense RNA results in a endogenous gene that reduce expression of the same or can switch off, similar to that described for antisense approaches (Goring et al. (1991) Proc. Natl Acad Sci USA, 88: 1770-1774; Smith et al. (1990) Mol Gen Genet 224: 447-481; Napoli et al. (1990) Plant Cell 2: 279-289; Van der Krol et al. (1990) Plant Cell 2: 291-299). The introduced construct can do that represent the reduced gene in whole or in part. The Possibility of translation is not necessary.

The use of methods is also very particularly preferred such as gene regulation using double-stranded RNA ("double- stranded RNA interference "). Corresponding methods are the Known to those skilled in the art and described in detail (e.g. Matzke MA et al. (2000) Plant Mol Biol 43: 401-415; Fire A. et al (1998) Nature 391: 806-811; Where 99/32619; Where 99/53050; Where 00/68374; Where 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). To those in the procedures and methods described quotations expressly referred. Here is through simultaneous contribution of strand and counter strand a highly efficient suppression native genes.

The antisense strategy can be advantageous with one Ribozyme processes are coupled. Ribozymes are catalytically active RNA Sequences that are linked to the antisense sequences that Split target sequences catalytically (Tanner NK. FEMS Microbiol Rev. 1999; 23 (3): 257-75). This can make an anti-sense more efficient Increase strategy. Expression of ribozymes for reduction certain proteins are known to the person skilled in the art and for example described in EP-A1 0 291 533, EP-A1 0 321 201 and EP-A1 0 360 257. Suitable target sequences and ribozymes can be, for example, like by Steinecke (Ribozymes, Methods in Cell Biology 50, Galbraith et al eds Academic Press, Inc. (1995), 449-460), by secondary structure calculations of ribozyme and target RNA as well as by their interaction (Bayley CC et al., Plant Mol Biol. 1992; 18 (2): 353-361; Lloyd AM and Davis RW et al., Mol Gen Genet. 1994 Mar; 242 (6): 653-657). Are exemplary To call "hammerhead" ribozymes (Haselhoff and Gerlach (1988) Nature 334: 585-591). Preferred ribozymes are based on derivatives of Tetrahymena L-19 IVS RNA (US 4,987,071; US 5,116,742). Further Ribozymes with selectivity for an L119 mRNA can be selected (Bartel D and Szostak JW (1993) Science 261: 1411-1418).

In a further embodiment, reduction in the Target protein expression using nucleic acid sequences are brought about, which are complementary to the regulatory elements of the Target protein genes are, with these, a triple-helical structure educate and thus prevent gene transcription (Helene C (1991) Anticancer drug des. 6 (6): 569-84; Helene C et al. (1992) Ann NY Acad Sci 660: 27-36; Maher LJ (1992) Bioassays 14 (12): 807-815).

The expression cassettes according to the invention and those of them derived vectors can contain further functional elements.

• a) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Transformationseffizienz, des Expressionsortes oder -zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Gene kodierend für Reporter-Proteine (siehe auch Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1): 29-44) wie
  • - "green fluorescence protein" (GFP) (Chui WL et al., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques. 23(5): 912-8, 1997; Sheen et al. (1995) Plant Journal 8(5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA 93(12): 5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267-271; WO 97/41228).
  • - Chloramphenicoltransferase (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82: 5824-5828),
  • - Luziferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414; Ow et al. (1986) Science, 234: 856-859); erlaubt Bioluminescenzdetektion.
  • - β-Galactosidase, kodiert für ein Enzym für das verschiedenen chromogene Substrate zur Verfügung stehen.
  • - β-Glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6, 3901-3907) oder das uidA Gen, das ein Enzym für verschiedene chromogene Substrate kodiert.
  • - R-Locus Genprodukt: Protein, das die Produktion von Anthocyaninpigmenten (rote Färbung) in pflanzlichen Gewebe reguliert und so eine direkte Analyse der Promotoraktivität ohne Zugabe zusätzlicher Hilfsstoffe oder chromogener Substrate ermöglicht (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, lBth Stadler Genetics Symposium, 11: 263-282, 1988).
  • - β-Lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA 75: 3737-3741), Enzym für verschiedene chromogene Substrate (z. B. PADAC, eine chromogenes Cephalosporin).
  • - xylE Genprodukt (Zukowsky et al. (1983) Proc Natl Acad Sci USA 80: 1101-1105), Catecholdioxygenase, die chromogene Catechole umsetzen kann.
  • - Alpha-Amylase (Ikuta et al. (1990) Bio/technol. 8: 241-242).
  • - Tyrosinase (Katz et al. (1983) J Gen Microbiol 129: 2703-2714), Enzym, das Tyrosin zu DOPA und Dopaquinon oxidiert, die infolge das leicht nachweisbare Melanin bilden.
  • - Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126(3): 1259-1268), kann in der Calcium-sensitiven Bioluminescenzdetektion verwendet werden.
• b) Replikationsursprünge, die eine Vermehrung der erfindungsgemässen Expressionskassetten oder Vektoren in zum Beispiel E.coli gewährleisten. Beispielhaft seien genannt ORI (origin of DNA replication), der pBR322 ori oder der P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• c) Elemente zum Beispiel "Bordersequenzen", die einen Agrobakterien-vermittelte Transfer in Pflanzenzellen für die Übertragung und Integration ins Pflanzengenom ermöglichen, wie zum Beispiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.
• d) Multiple Klonierungsregionen (MCS) erlauben und erleichtern die Insertion eines oder mehrerer Nukleinsäuresequenzen.

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 coassettes according to the invention or of vectors or organisms derived therefrom. Examples include, but are not limited to:

• a) Reporter genes, which code for easily quantifiable proteins and ensure an assessment of the transformation efficiency, the place of expression or the point in time via self-color or enzyme activity. Genes coding for reporter proteins are very particularly preferred (see also Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 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; Sheen et al. ( 1995) Plant Journal 8 (5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94 (6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA 93 (12) : 5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267-271; WO 97/41228).
  • Chloramphenicol transferase (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82: 5824-5828),
  • - Luciferase (Millar et al., Plant Mol Biol Rep 1992 10: 324-414; Ow et al. (1986) Science, 234: 856-859); allows bioluminescence detection.
  • - β-galactosidase, encoded for an enzyme for which various chromogenic substrates are available.
  • β-glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or the uidA gene, which encodes an enzyme for various chromogenic substrates.
  • - R-Locus gene product: protein that regulates the production of anthocyanin pigments (red color) in plant tissue and thus enables a direct analysis of the promoter activity without the addition of additional auxiliaries or chromogenic substrates (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, lBth Stadler Genetics Symposium, 11: 263-282, 1988).
  • β-lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA 75: 3737-3741), enzyme for various chromogenic substrates (e.g. PADAC, a chromogenic cephalosporin).
  • - xylE gene product (Zukowsky et al. (1983) Proc Natl Acad Sci USA 80: 1101-1105), catechol dioxygenase, which can convert chromogenic catechols.
  • Alpha amylase (Ikuta et al. (1990) Bio / technol. 8: 241-242).
  • - Tyrosinase (Katz et al. (1983) J Gen Microbiol 129: 2703-2714), enzyme that oxidizes tyrosine to DOPA and dopaquinone, which consequently form the easily detectable melanin.
  • - Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268), can be used in calcium-sensitive bioluminescence detection.
• b) Origins of replication, which ensure an increase in the expression cassettes or vectors according to the invention in, for example, E. coli. Examples include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sambrook et al .: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• c) Elements, for example “border sequences”, which enable agrobacterium-mediated transfer into plant cells for the transfer and integration into the plant genome, such as, for example, the right or left border of the T-DNA or the vir region.
• d) Multiple cloning regions (MCS) allow and facilitate the insertion of one or more nucleic acid sequences.

Various ways are known to the person skilled in the art to achieve a expression cassette according to the invention. The production an expression cassette according to the invention for example by fusion of one of the promoters according to the invention (or of a functional equivalent or a functional equivalent Partly in accordance with SEQ ID NO: 1, 2 or 3 or a functional one Equivalent with a nucleotide sequence to be expressed, optionally a sequence coding for a transit peptide, preferably a chloroplast-specific transit peptide, which preferably between the promoter and the respective Nucleotide sequence is arranged, as well as a terminator or Polyadenylation signal. Common recombination and Cloning techniques such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as 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, there are also those under an expression cassette Understand constructions in which the promoter is used without it functional with a nucleic acid sequence to be expressed was linked, for example via a targeted homologue Recombination or a random insertion into a host genome becomes regulatory control over with it then functional linked nucleic acid sequences takes over and the transgenic Expression controls same. By inserting the promoter - to Example by homologous recombination - before one for one a certain polypeptide encoding nucleic acid is obtained Expression cassette according to the invention, the expression of controls certain polypeptides in the plant. Furthermore, the Insertion of the promoter also take place in such a way that antisense RNA the nucleic acid encoding a particular polypeptide is expressed. This expresses the particular polypeptide down regulated in plants or switched off.

Analogously, a transgene can also be expressed Nucleic acid sequence for example by homologous recombination behind the endogenous, natural promoter can be placed, resulting in a Expression cassette according to the invention receives the expression the transgenic nucleic acid sequence to be expressed in the Controls cotyledons of the plant embryo.

According to the invention are also vectors that described above Expression cassettes included. Vectors can be exemplary Plasmids, cosmids, phages, viruses or even agrobacteria.

Another object of the invention relates to transgenic Organisms transformed with at least one according to the invention Expression cassette or a vector according to the invention, and Cells, cell cultures, tissues, parts - such as in vegetable organisms leaves, roots etc. - or Propagation material derived from such organisms.

Under organism, parent or host organisms prokaryotic or eukaryotic organisms, such as Microorganisms or plant organisms understood. 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 that are used for infection of plants and thus for the transfer of the invention Cassettes are capable. Preferred microorganisms are those from the genus Agrobacterium and in particular from Art Agrobacterium tumefaciens.

Preferred yeasts are Candida, Saccharomyces, or Hansenula 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.

Preferred host or as transgenic organisms The main organisms are plants. Included in the Invention all genera and species higher and lower Plants of the plant kingdom. The tires are also included Plants, seeds, sprouts and seedlings, as well as thereof derived parts, propagation material and cultures, for example Cell cultures. Mature plants mean plants to any Developmental stage beyond the seedling. Seedling means a young, immature plant at an early stage of development.

Annual, perennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the production of transgenic Plants. The expression of genes is also advantageous in all ornamental plants, useful or ornamental trees, flowers, Cut flowers, shrubs or lawn. Exemplary but not restrictive May be mentioned angiosperms, bryophytes such as Hepaticae (liverwort) and Musci (moss); Pteridophytes like ferns, Horsetail and lycopods; Gymnosperms like 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 examples and not restricting the families of Rosaceae like Rose, Ericaceae like Rhododendrons and azaleas, Euphorbiaceae such as poinsettias and Croton, Caryophyllaceae like cloves, Solanaceae like petunias, Gesneriaceae like the African violet, Balsaminaceae like that Balsam, orchidaceae like orchids, 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 more.

Exemplary but not restrictive for flowering plants name the families of leguminosae such as pea, alfalfa and soy; Gramineae such as rice, corn, wheat; Solanaceae like tobacco and others more; the Umbelliferae family, especially the genus Daucus (especially the species carota (carrot)) and Apium (whole especially the species graveolens dulce (Selaria)) and others more; the Family of the Solanacea, especially the genus Lycopersicon, whole especially the species esculentum (tomato) and the genus Solanum, whole especially the species tuberosum (potato) and melongena (eggplant) and others; and the genus Capsicum, especially the species annum (pepper) and others; the Leguminosae family, especially the genus Glycine, especially the species max (Soybean) and others more; and the Cruciferae family, especially the genus Brassica, especially the species 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 family of Compositae, especially the genus Lactuca, especially the species Sativa (salad) and others.

The transgenic plants according to the invention are in particular selected from monocot crops, such as Cereals such as wheat, barley, millet, rye, triticale, maize, Rice or oats and sugar cane. Furthermore, the transgenic plants according to the invention in particular selected from dicotyledonous crops, such as Brassicacae such as rapeseed, cress, arabidopsis, cabbage or canola, Leguminosae such as soy, alfalfa, peas, alfalfa, beans 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 flax, cotton, hemp, flax, red pepper, carrot, Carrot, sugar beet and the various tree, nut and Grapevine species.

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

Plant organisms in the sense of the invention are still other photosynthetically active competent organisms, such as Example algae or cyanobacteria, as well as mosses.

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

The production of a transformed organism or one transformed cell requires that the corresponding DNA be in the corresponding host cell is introduced. For this operation, which is called transformation stands for a variety of Methods are available (see also Keown et al. 1990 Methods in Enzymology 185: 527-537). For example, DNA can be direct by microinjection or by bombardment with DNA-coated microparticles are introduced. The cell can also 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 others DNA-containing units such as minicells, cells, or lysosomes Liposomes are done. Electroporation is another suitable method for the introduction of DNA, in which the cells are reversible through a electrical impulse can be permeabilized.

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

In addition to these "direct" transformation techniques, a Transformation also by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes can be performed. These strains contain a plasmid (Ti or Ri plasmid) which is based on the plant is transmitted after agrobaterium infection. A part this plasmid, called T-DNA (transferred DNA), is inserted into the Plant cell genome integrated.

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

The introduction of an expression cassette according to the invention in Cells, preferably in plant cells, can be beneficial under Using vectors can be realized.

In an advantageous embodiment, the introduction of Expression cassette realized using plasmid vectors. Prefers are such vectors that provide stable integration of the Enable expression cassette into the host genome.

In the case of injection or electroporation of DNA into plant cells are no special requirements for the used Plasmid. Simple plasmids like the pUC series can be used. Should whole plants from the transformed cells are regenerated, so it is required that there is an additional selectable marker gene on the plasmid located.

Transformation techniques are different for different monocots and dicotyledonous plant organisms. Also stand different possible plasmid vectors for the introduction of foreign ones Genes available in plants that are usually one Origin of replication for 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 using a suitable Restriction interface will be introduced. 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 serve the Check cloning step.

Transformed cells d. H. those that have the introduced DNA can be integrated into the DNA of the host cell untransformed can be selected if a selectable Marker is part of the DNA introduced. Can be used as a marker exemplary each gene act that resistance to Can confer antibiotics or herbicides. Transformed cells, who express such a marker gene are able to Presence of concentrations of an appropriate antibiotic or survive herbicides that are an untransformed wild type kill. Example are the bar gene that resistance to that Herbicide gives phosphinothricin (Rathore KS et al., Plant Mol Biol. 1993 Mar; 21 (5): 871-884), the nptII gene that resistance imparts resistance to kanamycin, the hpt gene Hygromycin confers, or the EPSP gene, resistance to the herbicide Gives glyphosate.

Depending on the method of DNA introduction, additional genes can be found on the Vector plasmid may be required. If Agrobacteria are used, so the expression cassette is in special plasmids integrate, either into an intermediate vector (English: shuttle or intermediate vector) or a binary vector. If for example a Ti or Ri plasmid is to be used for transformation, is at least the right boundary, but mostly the right one and the left boundary of the Ti or Ri plasmid T-DNA as flanking region with the expression cassette to be inserted connected. Binary vectors are preferably used. binary Vectors can be found in both E.coli and Agrobacterium replicate. They usually contain a selection marker gene and one Left or polylinker flanked by the right and left T-DNA Border sequence. You can go straight to Agrobacterium can be transformed (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The selection marker gene allows selection transformed Agrobacteria and is for example the nptII gene that confers resistance to kanamycin. In this case as Host organism that acts as Agrobacterium should already be one Contain plasmid with the vir region. This is for the Transfer of T-DNA to the plant cell required. Such a transformed Agrobacterium can be used for transformation plant 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., EMBO J. 4 (1985), 277-287). Various 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 herbal explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes co-cultivated. Starting from infected Plant material (e.g. leaf, root or stem parts, but also Protoplasts or suspensions of plant cells) can be whole plants using a suitable medium that for example Antibiotics or biocides for selection of transformed cells can contain, be regenerated. The plants obtained can then on the presence of the introduced DNA, here the expression cassette according to the invention. Once the DNA is integrated into the host genome is the corresponding genotype usually stable and the corresponding insertion is also in the successor generations. Usually contains the integrated expression cassette a selection marker that the transformed plant becomes resistant to a biocide (for Example a herbicide), a metabolism inhibitor such as 2-DOG or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin, etc. The selection marker allows the selection of transformed cells from untransformed ones (McCormick et al., Plant Cell Reports 5 (1986), 81-84). The Plants obtained can be grown and crossed in the usual way become. Two or more generations should be cultivated to ensure that genomic integration is stable and is heritable.

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) described. Preferably, the construct to be expressed is in one Vector cloned that is suitable for Agrobacterium tumefaciens transform, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711).

Once a transformed plant cell has been made, a whole plant using the skilled person known methods can be obtained. Here you go as an example from callus cultures. From these still undifferentiated Cell masses can cause sprout and root formation in a known manner be induced. The sprouts obtained can be planted out and be bred.

The effectiveness of expression of the transgenic expressed Nucleic acids can, for example, in vitro Shoot meristem propagation using one of those described above Selection methods are determined.

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

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

Another object of the invention relates to the use of Transgenic organisms according to the invention described above and the cells, cell cultures, parts derived from them - such as Example with transgenic plant organisms roots, leaves etc. -, 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 coassettes or vectors described above and this expression cassette contains one or more structural genes which code for the desired fine chemical or catalyze the biosynthesis of the desired fine chemical, 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 are grown and isolated from the host organisms or from the growth medium 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 Immunol. 1999; 236: 275-92. sequences

Examples General methods

The chemical synthesis of oligonucleotides can, for example, in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The in Performed within the scope of the present invention Cloning steps such as B. restriction splits, Agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, Propagation of phages and sequence analysis of recombinant DNA will be as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The Recombinant DNA molecules are sequenced using a Laser fluorescence DNA sequencer from ABI using the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467).

example 1 Isolation of genomic DNA from Arabidopsis thaliana (CTAB method)

For the isolation of genomic DNA from Arabidopsis thaliana, approx. 0.25 g leaf material of young plants in the vegetative stage are ground into fine powder in liquid nitrogen. The pulverized plant material is put together with 1 ml of 65 ° C warm CTAB I buffer (CTAB: hexadecyltrimethylammonium bromide, also called cetyltrimethylammonium bromide; Sigma cat.-no .: H6269) and 20 µl β-mercaptoethanol in a pre-warmed second mortar and after complete Homogenization, the extract is transferred to a 2 ml Eppendorf tube and incubated for 1 h at 65 ° C with regular, careful mixing. After cooling to room temperature, the mixture is extracted with 1 ml of chloroform / octanol (24: 1, shaken out with 1 M Tris / HCl, pH 8.0) by slow inverting and for phase separation for 5 min at 8,500 rpm (7,500 × g) and room temperature centrifuged. The aqueous phase is then extracted again with 1 ml of chloroform / octanol, centrifuged and mixed thoroughly by inverting with 1/10 volume of CTAB II buffer preheated to 65 ° C. Then the mixture is mixed carefully with 1 ml of chloroform / octanol mixture (see above) and centrifuged for 5 min at 8,500 rpm (7,500 × g) and room temperature to separate the phases again. The aqueous lower phase is transferred to a fresh Eppendorff tube and the upper organic phase is centrifuged again in a fresh Eppendorff tube for 15 min at 8,500 rpm (7,500 × g) and room temperature. The resulting aqueous phase is combined with the aqueous phase from the previous centrifugation step and the entire batch is mixed with exactly the same volume of preheated CTAB III buffer. This is followed by incubation at 65 ° C until the DNA precipitates in flakes. This can take up to 1 h or can be done by incubating at 37 ° C overnight. The sediment resulting from the subsequent centrifugation step (5 min. 2000 rpm (500 × g), 4 ° C.) is mixed with 250 μl of CTAB IV buffer preheated to 65 ° C. and for at least 30 min or until the sediment has completely dissolved incubated at 65 ° C. The solution for precipitating the DNA is then mixed with 2.5 volumes of ice-cold ethanol and incubated for 1 h at -20 ° C. Alternatively, the batch can be mixed with 0.6 volumes of isopropanol and centrifuged immediately for 15 min at 8,500 rpm (7,500 × g) and 4 ° C without further incubation. The sedimented DNA is washed twice with 1 ml of 80% ice-cold ethanol by inverting the Eppendorff tube, centrifuged again after each washing step (5 min. 8,500 rpm (7,500 × g), 4 ° C) and then for approx. 15 min air-dried. Finally, the DNA is resuspended in 100 µl TE with 100 µg / ml RNase and incubated for 30 min at room temperature. After a further incubation phase at 4 ° C overnight, the DNA solution is homogeneous and can be used for further experiments.
Solutions for CTAB:
Solution I (for 200 ml):
100 mM Tris / HCl pH 8.0 (2.42 g)
1.4 M NaCl (16.36 g)
20 mM EDTA (8.0 ml of 0.5 M stock solution)
2% (w / v) CTAB (4.0 g)
Before use, add fresh: 2% β-mercaptoethanol (20 µl for 1 ml solution I).
Solution II (for 200 ml):
0.7 M NaCl (8.18 g)
10% (w / v) CTAB (20 g)
Solution III (for 200 ml):
50 mM Tris / HCl pH 8.0 (1.21 g)
10 mM EDTA (4 ml 0.5 M of 0.5 M stock solution)
1% (w / v) CTAB (2.0 g)
Solution IV (high-salt TE) (for 200 ml):
10 mM Tris / HCl pH 8.0 (0.242 g)
0.1 mM EDTA (40 µl 0.5 M stock solution)
1 M NaCl (11.69 g)
Chloroform / octanol (24: 1) (for 200 ml)
192 ml chloroform
8 ml octanol
The mixture is shaken twice with 1 M TrisHCl pH 8.0 and stored away from light.

Example 2 tobacco transformation

The tobacco was transformed via infection with Agrobacterium tumefaciens. according to the method developed by Horsch (Horsch et al. (1985) Science 227: 1229-1231). All for Constructs used were based on the Freeze / thaw method (repeated thawing and freezing) in Agrobacterium tumefaciens transformed. The desired construct Colonies containing Agrobacterium were grown on mannitol / glutamate medium with 50 µg / ml kanamycin, 50 µg / ml ampicilin and 25 µg / ml rifampicin selected.

For the transformation of tobacco plants (Nicotiana tabacum L. cv. Samsun NN) 10 ml of a grown under selection Centrifuged overnight culture from Agrobacterium tumefaciens, the The supernatant was discarded and the bacteria in the same volume Antibiotic-free medium resuspended. In a sterile Petri dishes became leaf disks of sterile plants (diameter approx. 1 cm) bathed in this bacterial solution. Then the Leaf disks in petri dishes on MS medium (Murashige and Skoog (1962) Physiol Plant 15: 473ff.) With 2% sucrose and 0.8% Bacto- Designed agar. After 2 days of incubation in the dark at 25 ° C they were on MS medium with 100 mg / l kanamycin, 500 mg / l claforan, 1 mg / l benzylaminopurine (BAP), 0.2 mg / l naphthylacetic acid (NAA), 1.5% glucose and 0.8% Bacto agar transferred and the cultivation (16 hours light / 8 hours dark) continued. growing Rungs were placed on hormone-free MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto agar transferred.

Example 3 Studies on the suitability of putative ferredoxin (pFD) promoter a) Cloning of the pFD promoter from Arabidopsis thaliana

The putative ferredoxin promoter was amplified by PCR from genomic Arabidopsis thaliana DNA with the primers pWL35 and pWL36. The primer pWL35 begins with the SalI and EcoRI restriction sites highlighted in bold immediately before the coding region of the pFD gene. The primer pWL36 begins with the SalI and Asp718 restriction sites highlighted in bold.


Reaction:
1 µl genomic Arabidopsis DNA (approx. 250 ng)
5 µl Tth polymerase (2 U / µl)
3 µl Mg (OAc) ₂ (25 mM, final conc. 1.5 mM Mg ²⁺ )
15.2 µl 3.3 x buffer
4 µl dNTPs (2.5 mM each, Takara, final concentration: 200 µM each)
24.3 µl H ₂ O
PCR conditions:
1 cycle with 3 min. at 95 ° C
10 cycles with 94 ° C for 10 sec, 50 ° C for 20 sec and 72 ° C for 1 min.
20 cycles with 94 ° C for 10 sec, 65 ° C for 20 sec and 72 ° C for 1 min.
1 cycle at 72 ° C for 5 min.,
then cooling to 4 ° C until further processing.

b) Construction of the pFD promoter-GUS expression cassette

The pFD promoter PCR product was transformed into the vector pCRII (Invitrogen) cloned and then by means of the primer pair introduced SalI restriction sites isolated and cleaned by gel electrophoresis. For the fusion with the CIS gene was the approximately 850 bp pFD promoter fragment in the SalI-restricted binary vector pBI101.2 (Clontech Inc.) cloned and the orientation of the fragment was then determined using Restriction analyzes using the endonucleases BglII and BamHI verified. The resulting plasmid pFD :: GUS was described in Transformed tobacco. The tobacco plants produced were pFD: GUS called.

c) Construction of the pFD promoter nptII expression cassette

• 1. The putative ferredoxin promoter was amplified by PCR from genomic Arabidopsis thaliana DNA. The restriction sites EcoRI and NcoI were added via the primers.
  
  
  

Reaction:
37.5 µl H ₂ O
5.0 µl 10X reaction buffer (final concentration Mg ²⁺ 1.5 mM)
4.0 µl dNTP mix (2.5 mM each)
1.0 µl primer pFD1 (10 µM)
1.0 µl primer pFD2 (10 µM)
0.5 µl Taq polymerase (Takara, 2 U / µl)
1.0 µl genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle with 3 min. at 94 ° C
10 cycles with 94 ° C for 10 sec, 48 ° C for 20 sec and 72 ° C for 1 min
25 cycles with 94 ° C for 10 sec, 65 ° C for 20 sec and 72 ° C for 1 min
1 cycle at 72 ° C for 5 min.

The PCR product was transformed into the plasmid pCRII (Invitrogen) subcloned. The plasmid pCAMBIA 2300 (CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia; GenBank Acc.-No: AF234315; Binary vector pCAMBIA-2300, complete sequence; Hajdukiewicz P et al. (1994) Plant Mol Biol 25 (6): 989-994) was cut with EcoRI / NcoI and the pFD promoter fragment as an EcoRI / NcoI fragment from the pCRII plasmid cloned into this vector. The 35S Promoter removed from the cambia vector. The resulting plasmid was designated as pFD promoter: NPTII and in tobacco transformed.

d) Results of the CIS analysis of the transgenic tobacco plants

In the context of histochemical studies, transgenic pFD:: -GUS tobacco plants showed a strong GUS staining in 'source' leaves and a weak GUS staining in the tissues of all flower organs. A strong staining in the root tissue was only observed in in vitro plants whose roots were exposed to the light. Callus growth was induced using leaf disks punched out from plants identified as pFD :: GUS positive. The callus tissue and the plant shoots that developed from it showed a GUS staining, which in its intensity corresponds to the GUS staining of CaMV35S :: GUS (in pCambia 1304; CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia; GenBank Acc.-No. : AF234300, Binary vector pCAMBIA-1304, complete sequence, Hajdukiewicz P et al. (1994) Plant Mol. Biol. 25 (6): 989-994) (1994)) was comparable to transgenic plants. In the table below (Tab. 3) the data for the quantification of the GUS activity in the anthers (anthers) and 'source' leaves of selected transgenic pFD :: GUS tobacco plants were compiled. Table 3 Quantification of GUS activity in the anthers and 'source' leaves of selected transgenic pFD :: GUS tobacco plants

The anthers of the mature flowers do not branch any promoter activity. It is weakly pronounced when the flowers are closed.

e) Results of the analysis of the kanamycin resistance of the transgenic tobacco plants

For the study of the pFD promoter-mediated mediation of Resistance to kanamycin was the pFD promoter: NPTII plasmid in Transformed tobacco. The selective regeneration of the Tobacco plants were made on kanamycin (100 mg / l). The one from itself developing bud buds regenerated plants contained that Kanamycin, which showed that the pFD promoter expresses the NPTII gene has and thus made the selection possible. The results demonstrate that the isolated nucleic acid sequence is the desired one has advantageous promoter properties, d. H. she shows one Promoter activity that is suitable for effective selection markers express and has little activity in the pollen. The activity in the anthers is usually less than 10% of the activity in the source sheets.

f) Preparation of deletion variants of the pFD promoter

Another variant of the pFD promoter is the deletion pFDshort (pFds). For this purpose, the section from base pair 137 to 837 of the pFD promoter was amplified using the following primers:


Reaction:
37.5 µl H ₂ O
5.0 µl 10X reaction buffer ("genomic PCR")
4.0 µl dNTP mix (2.5 mM each)
2.2 µl 25 mM Mg (OAc) 2 (final concentration 1.1 mM)
1.0 µl primer pFD3 (10 µM)
1.0 µl primer pFD5 (10 µM)
0.5 µl Pfu-turbo polymerase mix
1.0 µl genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle with 5 min. at 95 ° C
25 cycles with 94 ° C for 30 sec, 50 ° C for 60 sec and 72 ° C for 1 min.
1 cycle at 50 ° C for 60 sec, 72 ° C for 10 min.,
then cooling to 4 ° C until further processing

The primers contained recognition sequences for the restriction enzyme BamHI. After cleavage of the PCR product with BamHI, it was ligated into the plasmid pGUSINT37 (see above), which was also cut by BamHI and dephosphorylated. The resulting construct pFDsGUSINT was used to bombard tobacco leaves with the Biolistics (BioRad). Microcarriers (25 µg gold, Heraeus 0.3-3 µm) were treated with 10 µg plasmid DNA, 2.5 M CaCl ₂ , and 0.1 M spermidine, washed with alcohol and under a vacuum of 26 inches and a pressure shot at 1100 psi on the sheets lying on MS medium. The explants were then incubated for 24 h in MS medium with 2% sucrose and then histochemically stained with X-gluc. Blue spots showed the activity of the promoter.

g) Fusion of the pFds promoter with the NPTII gene

As a BamHI fragment, the pFDs promoter is made from pFDsGUSINT cut out and smoothed its ends with Klenow "Fill-In". The fragment obtained is placed in front of the NPTII gene of the plasmid pSUNSNPTII (SEQ ID NO: 24), EcoRV cut and dephosphorylated, cloned. The plasmid pSUNSNPTII is a derivative of the Plasmids pSUN3 (SEQ ID NO: 23), which is next to a nosP / Pat cassette still contains a promoterless NPTII gene. With this construct it is possible for promoters to express their ability Test NPTII. In parallel, the selection for phosphinothricin containing medium.

The resulting plasmid pSun5FdsNPTII is in tobacco transformed. Regenerated and selected shoots showed that the pFDs Promoter allows selection for NPTII.

Example 4 Studies on the suitability of the ferredoxin NADPH Oxidoreductase (FNR) promoter a) Cloning of the FNR promoter from Arabidopsis thaliana

The putative promoter region of the FNR gene was amplified from genomic DNA using the oligonucleotide primers L-FNRara and R-FNRara, leaving out the ATG start codon of the FNR gene and maintaining four putative stop codons of the upstream open reading frame. Using the primers L-FNRara and R-FNRara, the FNR promoter was amplified as a 635 bp fragment corresponding to the section of clone K2A18.15 from position 69493 to position 70127 (both nucleotides included) by means of PCR from genomic Arabidopsis thaliana DNA. The primer L-FNRara begins with the SalI and BamHI restriction sites highlighted in bold and is located upstream of the four stop codons of the gene upstream of the FNR promoter. The primer R-FNRara begins with the SalI and XbaI restriction sites highlighted in bold immediately before the ATG start codon of the FNR gene.

A so-called 'touchdown' PCR protocol using the 'Advantage Genomic Polymerase Mix' (Clontech Laboratories, Inc. Catalog No. # 8418-1) was used to amplify the FNR promoter. The above-mentioned polymerase mix contains a thermostable DNA polymerase from Thermus thermophilus (Tth DNA polymerase), mixed with a smaller portion of the "Vent Proofreading 3'-5 'polymerase", and the Tth Start antibody, the "hot-start" PCR allows.
Reaction:
3 6.8 µl H ₂ O
5 µl 10X reaction buffer ("genomic PCR")
1 µl dNTP mix (2.5 mM each)
2.2 µl 25 mM Mg (OAc) ₂ (final concentration 1.1 mM)
1 µl primer L-FNR ara (10 µM)
1 µl primer R-FNR ara (10 µM)
1 µl 50 × polymerase mix
2 µl genomic Arabidopsis DNA (approx. 500 ng)
PCR conditions:
1 cycle with 1 min. at 94 ° C
10 cycles with 94 ° C for 30 sec and 70 ° C for 3 min.
32 cycles with 94 ° C for 30 sec and 65 ° C for 3 min.
1 cycle at 65 ° C for 4 min.,
then cooling to 4 ° C until further processing.

b) Construction of the FNR promoter-GUS expression cassette

The FNR promoter PCR product became electrophoretic after gel Separation and purification from the gel with the Quiagen PCR purification kit via TA cloning into the vector pCRII (Invitrogen) cloned. The promoter fragment was then extracted from the resulting plasmid pATFNR1 by means of the primer pair introduced XbaI and BamHI restriction sites by digestion isolated with XbaI / BamHI and purified by gel electrophoresis. For the fusion with the GUS gene became the approximately 600 bp FNR- Promoter fragment in the XbaI / BamHI restricted binary vector pBI101 cloned. The correct insertion of the right fragment in the resulting plasmid pATFNR-Bi was then used a restriction analysis using the endonucleases EcoRV Verified. The plasmid pATFNR-Bi was used for tobacco transformation used.

c) Construction of the FNR promoter-PAT expression cassette

For the study of the FNR promoter-mediated mediation of Resistance to phosphinothricin was the FNR promoter as SalI Fragment from the plasmid pATFNR1 in front of the phosphinothricin Resistance gene in the SalI cut plasmid pSUN3PatNos (SEQ ID NO: 25) cloned.

d) Results of the CIS analysis of the transgenic tobacco plants Qualitative data

Transgenic FNR :: GUS plants showed strong GUS expression in all green fabrics, especially in 'source' leaves with petioles and internodes as well as all flower organs fully developed Flowers (ovary, stigma, sepals and petals) with the exception of Pollen that showed no GUS activity; a small one Staining intensity was detected in anthers. The first analysis of Leaf disks from 80 in vitro plants showed 70 plants one strong GUS staining with little variation in staining intensity between the individual plants. That was used as a hint evaluated that the FNR promoter contains an element that for limited position effects. In the tissue crops was the GUS activity of the FNR :: GUS plants is significantly lower than that the TPT :: GUS plants.

Seed material (F1) from lines FNR 13, FNR 45 and FNR 28 were analyzed for their GUS activity. It turned out that neither in resting seeds nor in seedlings (3.5 Days after sowing) GUS activity could be detected. In later seedling stages (6 and 10 days after sowing) strong GUS activity in cotyledons and in the upper part of the Seedling axis detected, whereas no GUS staining in the Roots was found. In seedlings that are grown in the dark GUS activity was restricted to the cotyledons and was overall less than that of light-sprouted seedlings.

Quantitative data

The quantitative investigation of GUS activity in FNR :: GUS transgenic tobacco plants (transformed with plasmid pATFNR-Bi) was analyzed on the first fully developed leaves of tobacco plants 21 days after the transfer from the tissue culture to the greenhouse. The data correspond to the mean of four independent measurements. Table 4 Quantification of GUS activity in leaf material from selected transgenic FNR :: GUS tobacco plants (transformed with plasmid pATFNR-Bi)

e) Results of the analysis of the phosphinothricin resistance of the transgenic tobacco plants

The plasmid pSUN3FNRPat was constructed using the Agrobacterium tumefaciens strain EHA101 for tobacco transformation, as under 3. described, used. The selective regeneration of the Tobacco plants were made on the one hand on phosphinothricin (5 mg / l) and on the other hand, to check for kanamycin (100 mg / l). 97% of Explants that are under Kanamycin and 40% of the explants that selected under phosphinothric pressure, developed Shoot buds. The plants regenerated from it contained both the kanamycin as well as the phosphinothricin gene which showed that the FNR promoter the phosphinothricin acetyltransferase gene has expressed and thus made the selection possible. The results demonstrate that the nucleic acid sequence isolated the has desired advantageous promoter properties, d. H. she shows a promoter activity that is suitable, selection markers to express effectively and has no activity in the pollen.

Example 5 Studies on the suitability of triose phosphate Translocator (TPT) promoter a) Cloning of the TPT promoter from Arabidopsis thaliana

The putative promoter region of the Arabidopsis thaliana TPT gene was isolated by amplification with the oligonucleotide primers L-TPTara and R-TPTara, thereby leaving out the ATG start codon of the TPT gene. Using the primers L-TPTara and R-TPTara, the TPT promoter was amplified as a 2039 bp fragment by means of PCR from Arabidopsis thaliana genomic DNA. The L-TPTara primer begins with the SalI and BamHI restriction sites highlighted in bold. The primer R-TPTara begins with the AatII, SalI and XbaI restriction sites highlighted in bold immediately before the ATG start codon of the TPT gene.

A so-called 'touchdown' PCR protocol using the 'Advantage Genomic Polymerase Mix' (Clontech Laboratories, Inc. Catalog No. # 8418-1) was used to amplify the TPT promoter. The above-mentioned polymerase mix contains a thermostable DNA polymerase from Thermus thermophilus (Tth DNA polymerase), mixed with a smaller proportion of the 'vent proofreading 3'-5' polymerase ', and the Tth Start antibody, the "hot-start" PCR allows.
Reaction:
36.8 µl H ₂ O
5 µl 10X reaction buffer ("genomic PCR")
1 µl dNTP mix (2.5 mM each)
2.2 µl 25 mM Mg (OAc) ₂ (final concentration 1.1 mM)
1 µl primer L-FNR ara (10 µM)
1 µl primer R-FNR ara (10 µM)
1 µl 50 × polymerase mix
2 µl genomic Arabidopsis DNA (approx. 500 ng)
PCR conditions:
1 cycle with 1 min. at 94 ° C
10 cycles with 94 ° C for 30 sec and 70 ° C for 3 min.
32 cycles with 94 ° C for 30 sec and 65 ° C for 3 min.
1 cycle at 65 ° C for 4 min., Then cooling to 4 ° C until further processing.

b) Construction of the TPT promoter-GUS expression cassette

The TPT promoter PCR product became electrophoretic after gel Separation and purification from the gel with the Quiagen PCR purification kit via TA cloning into the vector pCRII (Invitrogen) cloned. The promoter fragment was then extracted from the resulting plasmid pATTPT by means of the primer pair introduced SalI and XbaI restriction interfaces isolated and cleaned by gel electrophoresis. For the fusion with the CIS gene was the approximately 2.2 kb TPT promoter fragment in the SalI / XbaI -restricted binary vector pBI101 cloned. The correct one Insertion of the correct fragment in the resulting plasmid pATTPT-Bi was then based on a restriction analysis Use of the endonucleases HindIII verified. The plasmid pATTPT-Bi was used for tobacco transformation.

b) Construction of the TPT promoter-PAT expression cassette

For the study of the TPT promoter-mediated mediation of Resistance to phosphinothricin was the TPT promoter as SalI Fragment from the plasmid pATTPT before the phosphinothricin Resistance gene cloned into the SalI-cut plasmid pSUN3PatNos. The resulting plasmid pSUN3TPTPat was cut using the Agrobacterium tumefaciens strain EHA101 for tobacco transformation used. The selective regeneration of the tobacco plants was done on the one hand on phosphinothricin (5 mg / l) and on the other hand to check for kanamycin (100 mg / l).

c) Results of the CIS analysis of the transgenic tobacco plants Qualitative data

Transgenic TPT :: GUS plants showed strong GUS expression in all green tissues, especially in 'source' leaves, here especially in the trichomes and flower organs young and full developed flowers. The GUS activity was in the field of flowers strongest in the ovaries and stigma; the coloring of the chalice and petals were a little weaker. In the anthers was the CIS Least activity and difficult to detect.

In the first analysis of leaf disks of 80 in In vitro plants, 22 plants showed no staining at all and 22 plants a strong GUS stain after only three hours of staining. The other plants showed a wide range of different intensities GUS stains on individual plants. In the In tissue culture plants, the GUS activity of the TPT :: GUS plants was clear stronger than that of the FNR :: GUS plants. Seed material (F1) the Lines TPT 55 and TPT 60 were checked for their GUS activity analyzed. It was found that both in resting seeds as well a strong one in seedlings (3.5 days after sowing) GUS activity could be detected. In later Seedling stages (6 and 10 days after sowing) became the strongest GUS activity in cotyledons and in the upper area of the seedling axis detected and a weaker GUS staining in the roots. Seedlings that were grown in the dark showed an unchanged GUS staining.

Quantitative data

The quantitative study of GUS activity in TPT :: GUS transgenic tobacco plants (transformed with plasmid pAT-TPT-Bi) was analyzed on the first fully developed leaves of tobacco plants 19 days after the transfer from the tissue culture to the greenhouse. The data correspond to the mean of four independent measurements. Table 5 Quantification of GUS activity in leaf tissue of selected transgenic TPT :: GUS tobacco plants (transformed with plasmid pAT-TPT-Bi). WT2: control values of untransformed wild-type plants

e) Results of the analysis of the phosphinothricin resistance of the transgenic tobacco plants

The plasmid pSUN3TPTPat was constructed using the Agrobacterium tumefaciens strain EHA101 for tobacco transformation, as under 3. described, used. The selective regeneration of the Tobacco plants were made on the one hand on phosphinothricin (5 mg / l) and on the other hand, to check for kanamycin (100 mg / l). 97% of Explants that are under Kanamycin and 70% of the explants that selected under phosphinothric pressure Shoot buds. The plants regenerated from it contained both the kanamycin as well as the phosphinothricin gene which showed that the TPT promoter the phosphinothricin acetyltransferase gene has expressed and thus made the selection possible. The results demonstrate that the nucleic acid sequence isolated the has desired advantageous promoter properties, d. H. she shows a promoter activity that is suitable for selection markers to express effectively and has no activity in the pollen.

Comparative Example 1 Studies on the suitability of the ubiquitin promoter a) Cloning of the ubiquitin promoter from Arabidopsis thaliana

The ubiquitin promoter was amplified by PCR from genomic Arabidopsis thaliana DNA with the primers ubi5 and ubi3.


Reaction:
37.5 µl H ₂ O
5 µl 10X reaction buffer ("genomic PCR")
4 µl dNTP mix (2.5 mM each)
2.2 µl 25 mM Mg (OAc) ₂ (final concentration 1.1 mM)
1 µl primer ubi3 (10 µM)
1 µl primer ubi5 (10 µM)
0.5 µl Pfu-turbo polymerase mix
1 µl genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle with 5 min. at 94 ° C
25 cycles at 94 ° C for 30 sec, 52 ° C for 1 min. and 72 ° C for 1 min.
1 cycle at 52 ° C for 1 min. and 72 ° C for 10 min.,
then cooling to 4 ° C until further processing.

The resulting PCR fragment was used as a HindIII / BamHI fragment in cloned the plasmid pGUSINT37 cut with HindIII / BamHI (pUBI42GUS) and verified by sequence analysis.

b) Cloning of the ubiquitin promoter before the PAT gene

Supported for the study of the ubiquitin promoter The resistance to phosphinothricin was mediated by the ubiquitin Promoter as a BamHI / HindIII fragment in front of the phosphinothricin Resistance gene in the BamHI / HindIII cut plasmid pSUN3PatNos cloned. The resulting plasmid pSUN3UBIPat was created under Use of the Agrobacterium tumefaciens strain EHA101 for Tobacco transformation used. The selective regeneration of the Tobacco plants were made on the one hand on phosphinothricin (5 mg / l) and on the other hand, to check for kanamycin (100 mg / l).

c) Results of the analysis of the phosphinothricin resistance of the transgenic tobacco plants

In contrast to the selection for kanamycin, which was normal, could not call or calli with selection for phosphinothricin Rung can be obtained. The ubiquitin promoter is not like this suitable for the expression of a selective marker for the Agrobacterium tumefaciens -mediated gene transfer with subsequent Regeneration of tissues.

Comparative Example 2 Studies on the suitability of squalene Synthase (SQS) promoters a) Cloning of the squalene synthase (SQS) promoter from Arabidopsis thaliana

The squalene synthase promoter was amplified by PCR from genomic Arabidopsis thaliana DNA with the primers sqs5 and sqs3.


reaction
37.5 µl H ₂ O
5 µl 10X reaction buffer ("genomic PCR")
4 µl dNTP mix (2.5 mM each)
2.2 µl 25 mM Mg (OAc) ₂ (final concentration 1.1 mM)
1 µl primer sqs3 (10 µM) (10 µM)
1 µl primer sqs5 (10 µM)
0.5 µl Pfu-turbo polymerase mix
1 µl genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle with 5 min. at 94 ° C
25 cycles at 94 ° C for 30 sec, 52 ° C for 1 min. and 72 ° C for 1 min.
1 cycle at 52 ° C for 1 min. and 72 ° C for 10 min.,
then cooling to 4 ° C until further processing.

The resulting PCR fragment was used as an XbaII / BamHI fragment in cloned the plasmid pGUSINT37 cut with XbaII / BamHI (pSQSPGUS) and verified by sequence analysis.

b) Cloning of the squalene synthase promoter in front of the PAT gene

Supported for the study of the squalene synthase promoter Mediation of resistance to phosphinothricin was the Squalene synthase promoter as a BamHI / SalI fragment in front of the Phosphinothricin resistance gene in the BamHI / SalI cut plasmid pSUN3PatNos cloned. The resulting plasmid was pSUN3SQSPat using the Agrobacterium tumefaciens strain EHA101 for Tobacco transformation used. The selective regeneration of the Tobacco plants were made on the one hand on phosphinothricin (5 mg / l) and on the other hand to control for kanamycin (100 mg / l).

c) Results of the analysis of the phosphinothricin resistance of the transgenic tobacco plants

In contrast to the selection for kanamycin, which was normal, could not call or calli with selection for phosphinothricin Rung can be obtained. The ubiquitin promoter is not like this suitable for the expression of a selective marker for the Agrobacterium tumefaciens -mediated gene transfer with subsequent Regeneration of tissues.

Comparative Example 3 Testing the promoter activity of the ubiquitin and squalene synthase promoters Partikelkanone

In order to test the activity of the ubiquitin and squalene synthase promoter transiently, sterile tobacco leaves were bombarded with plasmid DNA of the plasmids pUBI42GUS, pSQSPGUS and pGUSINT37 with the particle gun "Biolistics" from BioRad. Microcarriers (25 µg gold, Hereus 0.3-3 µm) were treated with 10 µg plasmid DNA, 2.5 M CaCl ₂ , and 0.1 M spermidine, washed with alcohol and under a vacuum of 26 inches and a pressure shot at 1100 psi on the leaves lying on MS agar medium. The explants were then incubated for 24 h in MS medium with 2% sucrose and then histochemically stained with X-gluc.

In contrast to the comparative construct pGUSINT37, where the GUS gene was expressed under the control of the 35S promoter, the ubiquitin promoter and the squalene synthase promoter showed only very few and very weak points with GUS staining. This shows that the activity of the ubiquitin and squalene synthase promoters is significantly weaker than that of the CaMV35S promoter. SEQUENCE LISTING

Claims (28)

1. Containing expression cassette for the transgenic expression of nucleic acids a) a promoter according to SEQ ID NO: 1, 2 or 3 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 transgenic nucleic acid sequence. 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 transgenically 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. 4. Expression cassette according to one of claims 1 to 3, wherein the nucleic acid sequence to be expressed transgenically selected is coding for selection markers from nucleic acids, Reporter genes, cellulases, chitinases, glucanases, Ribosome-inactivating proteins, Lysozyme, Bacillus thuringiensis Endotoxin, α-amylase inhibitor, protease inhibitors, lectins, RNAases, ribozymes, acetyl-CoA carboxylases, phytases, the 2S Albumin from Bertholletia excelsa, "antifreeze" proteins, Trehalose phosphate synthase, trehalose phosphate phosphatase, Trehalase, DREB1A factor, farnesyl transferases, ferritin, Oxalate oxidase, calcium-dependent protein kinases, calcineurins, Glutamate dehydrogenases, the N-hydroxylating, multifunctional cytochrome P-450, the transcriptional activator CBF1, Phytoendesaturases, polygalacturonases, Flavonoid-3'-hydroxylases, dihydroflavanol-4-reductases, chalconisomerases, Chalcone synthases, flavanone-3-beta-hydroxylases, flavone synthase II, branching enzyme Q, "Starch Branching" enzymes. 5. Expression cassette according to one of claims 1 to 4, wherein the nucleic acid sequence to be expressed transgenically selected is from the group of nucleic acid sequences with the gene bank Accession numbers U77378, AF306348, A19451, L25042, 578423, U32624, X78815, AJ002399, AF078796, AB044391, AJ222980, X14074, AB045593, AF017451, AF276302, AB061022, X72592, AB045592, AR123356. 6. Transgenic expression cassette according to one of claims 1 to 3 where the transgenic nucleic acid sequence to be expressed is selected from the group consisting of positive ones Selection markers, negative selection markers and factors grant a growth advantage. 7. Transgenic expression cassette according to claim 6, characterized marked that the selection marker is selected from the group consisting of proteins that are resistant to Antibiotics, metabolism inhibitors, herbicides or biocides to lend. 8. Transgenic expression cassette according to one of claims 6 or 7, characterized in that the selection marker one is selected from the group consisting of proteins Resistance to phosphinothricin, glyphosate, Bromoxynil, dalapon, 2-deoxyglucose-6-phosphate, tetracyclines, Ampicillin, kanamycin, G 418, neomycin, paromomycin, bleomycin, Zeocin, hygromycin, chloramphenicol, Sulfonylurea herbicides, imidazolinone herbicides. 9. The transgenic expression cassette according to any one of claims 6 to 8, characterized in that the selection marker is selected from phosphinothricin, 5-enolpyruvylshikimate-3-phosphate from the group Glyphosatoxidoreduktasen, dehalogenase, nitrilases, neomycin phosphotransferases, DOG ^R 1 genes, acetolactate synthases , Hygromycin phosphotransferases, chloramphenicol acetyl transferases, streptomycin adenylyl transferases, β-lactamases, tetA genes, tetR genes, isopentenyl transferases, thymidine kinases, diphthematoxin A, cytosine deaminase (codA), cytochrome P450Hogeno, genes, p2alogenase, halo. 10. Transgenic expression cassette according to one of claims 6 to 9, characterized in that the selection marker is encoded by nucleic acid sequences a) described by SEQ ID NO: 5 or 6, or b) described by or obtained in the sequences described by GenBank Acc.-No .: X17220, X05822, M22827, X65195, AJ028212, X17220, X05822 M22827, X65195, AJ028212, X63374, M10947, AX022822, AX1322820, E00622820, E01322820, E01322820, E01322820, E01322820, E01322820, E01322820, E01322820, E01322820, E01322820, E0106228, E0106228, E0106228, E010822820 AF080390, AF234316, AF080389, AF234315, AF234314, U00004, NC001140, X51514, AB049823, AF094326, X07645, X07644, A19547, A19546, A19545, I05376, I05373 AF34, AF34342929343429293434294 X65876, X51366, AJ278607, L36849, AB025109, AL133315. 11. Vectors containing an expression cassette according to one of the Claims 1 to 10. 12. A method for the transgenic expression of nucleic acids, characterized in that a nucleic acid sequence in functional linkage with a) a promoter according to SEQ ID NO: 1, 2 or 3 or b) a functional equivalent or equivalent fragment of a) which has essentially the same promoter activities as a), is expressed transgenically. 13. The method according to claim 12, characterized in that a) the nucleic acid sequence to be expressed is functionally linked to further genetic control sequences, or b) a used expression cassette contains additional functional elements, or c) a) and b) are given. 14. The method according to any one of claims 12 or 13, characterized in that the transgenic 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. 15. The method according to any one of claims 12 to 14, wherein the nucleic acid sequence to be expressed transgenically is selected from nucleic acids coding for selection markers, Reporter genes, cellulases, chitinases, glucanases, Ribosome inactivating proteins, lysozymes, Bacillus thuringiensis endotoxin, α-amylase inhibitor, protease inhibitors, lectins, RNAases, Ribozymes, acetyl-CoA carboxylases, phytases, the 2S albumin from Bertholletia excelsa, "antifreeze" proteins, Trehalose phosphate synthase, trehalose phosphate phosphatase, trehalase, DREB1A factor, farnesyl transferases, ferritin, oxalate oxidase, Calcium-dependent protein kinases, calcineurins, Glutamate dehydrogenases, the N-hydroxylating, multifunctional Cytochrome P-450, the transcriptional activator CBF1, Phytoendesaturases, polygalacturonases, flavonoid-3'-hydroxylases, Dihydroflavanol-4-reductases, chalconisomerases, Chalcon synthases, flavanone-3-beta-hydroxylases, flavone synthase II, Branching enzyme Q, "Starch Branching" enzyme. 16. The method according to any one of claims 12 to 15, wherein the nucleic acid sequence to be expressed transgenically is selected from the group of nucleic acid sequences with the gene bank Accession numbers U77378, AF306348, A19451, L25042, S78423, U32624, X78815, AJ002399, AF078796, AB044391, AJ222980, X14074, AB045593, AF017451, AF276302, AB061022, X72592, AB045592, AR123356. 17. A method for the selection of transformed organisms, characterized in that a nucleic acid sequence coding for a selection marker in a functional, transgenic linkage with a) a promoter according to SEQ ID NO: 1, 2 or 3, or b) a functional equivalent or equivalent fragment of a) which has essentially the same promoter activities as a), is introduced into an organism, the selection marker is expressed and a selection is carried out. 18. The method according to claim 17, characterized in that the Selection marker is selected from those in claims 6, 7, 8, 9 or 10 groups of selection markers. 19. Transgenic organism transforms with one Expression cassette according to claims 1 to 10 or a Vekor according Claim 11. 20. Transgenic organism according to claim 19 selected from the Group consisting of bacteria, yeasts, fungi, animal and vegetable organisms. 21. Transgenic organism according to one of claims 19 or 20 selected from the group consisting of arabidopsis, tomato, Tobacco, potatoes, corn, rapeseed, wheat, barley, sunflowers, Millet, beet, rye, oats, sugar beet, beans and Soy. 22. Cell cultures, parts or transgenic propagation material derived of a transgenic organism according to one of claims 19 until 21. 23. Use of a transgenic organism according to one of the Claims 19 to 21 or cell cultures derived therefrom, parts or transgenic propagation material according to claim 22 Manufacture of food, animal feed, seeds, pharmaceuticals or fine chemicals. 24. Use according to claim 23, wherein the fine chemicals Enzymes, vitamins, amino acids, sugar, saturated or unsaturated fatty acids, natural or synthetic flavor, Aroma or coloring are. 25. Use according to claim 23, wherein the pharmaceutical is a Antibody, enzyme or pharmaceutically active protein. 26. Process for the manufacture of pharmaceuticals or Fine chemicals in transgenic organisms according to one of claims 19 to 21 or cell cultures, parts or derived from these Transgenic propagation material according to claim 22, characterized characterized that the transgenic organism was bred and that desired pharmaceutical or fine chemical is isolated. 27. The method according to claim 26, wherein the fine chemicals are enzymes, Vitamins, amino acids, sugar, saturated or unsaturated Fatty acids, natural or synthetic taste, aroma or dyes. 28. The method of claim 26, wherein the pharmaceutical is a Antibody, enzyme or pharmaceutically active protein.