WO2001088164A1

WO2001088164A1 - Inducible elimination of dna sequences in plants

Info

Publication number: WO2001088164A1
Application number: PCT/DE2001/000780
Authority: WO
Inventors: Klaus Düring
Original assignee: Mpb Cologne Gmbh
Priority date: 2000-05-19
Filing date: 2001-02-28
Publication date: 2001-11-22
Also published as: AU2001246363A1; DE10024740A1

Abstract

The invention relates to a method for the controlled elimination of a desired DNA sequence in a host organism, preferably a transgenic plant, said method being based on a recombinase ligand-binding-domain fusion protein (Rec-LBD). The elimination of the desired DNA sequence only takes place after the expression of the active Rec-LBD by the activation of the inducible promoter, which controls the Rec-LBD coding gene, and the addition of the ligand which binds specifically to the Rec-LBD. The DNA sequence to be eliminated preferably codes for a marker gene, or acts as a transcription or translation stop sequence. The invention also relates to vectors and host organisms, preferably transgenic plants, which are suitable for use in said method.

Description

INDUCIBLE ELIMINATION OF DNA SEQUENCES IN PLANTS

The present invention relates to a method for the controlled elimination of a desired DNA sequence in a host organism, preferably a transgenic plant, characterized in that in step (I) the host organism with (a) the 5 'and 3' recombinations to be eliminated later DNA sequences flanked DNA sequence and (b) a ligand-binding-domain-recombinase-fusion protein (Rec-LBD) encoding these recombinant DNA sequences, which is transformed under conditions under the control of an inducible promoter to which the inducible promoter is repressed, the ligand specifically binding to Rec-LBD being absent; and in step (II) the DNA sequence to be eliminated is eliminated via the expression of Rec-LBD by activating the inducible promoter and the activation of Rec-LBD by adding the ligand which specifically binds to Rec-LBD at the desired point in time. In a preferred embodiment of the method according to the invention, the DNA sequence to be eliminated codes for a marker gene or acts as a transcription or translation stop sequence. The present invention also relates to the above sequences (a) and (b) containing vectors and host organisms, which are preferably transgenic plants.

The undirected integration of foreign DNA into plant cells is possible in principle. Compared to those used in human, mammalian or insect cell technologies for the targeted integration of DNA at specific locations of the genome or directed excision of the integrated DNA, they could tech ^¬ nologies are no established application-capable in plants. The removal of DNA in prokaryotes and in human, mammalian or insect cells, for example by using sequence-specific recombinases achieved. Initially, the corresponding integrated recombinase genes were placed under the control of inducible or tissue-specific promoters in order to be able to control the time or the location of the DNA elimination. However, control via an inducible or tissue-specific promoter has so far proved to be insufficiently flexible and controllable. In addition, the availability of ideally suitable promoters without significant restrictions in specificity is extremely low.

Alternatively, the approach of functional repression of the recombinase function was followed by the fusion of a ligand binding domain of a nuclear receptor to the recombinase. The Rec-LBD fusion protein turned out to be enzymatically represented, whereby the enzyme function could be restored by adding the ligand (EP-Bl 0 707 599). However, no complete repression can be obtained here either, for example due to protein cleavage at the fusion site by host-specific proteases, which can release the functional recombinase. This emerges from (Logie and Stewart, Proc.Natl.Acad.Sci.USA 92 (1995), 5940-5944).

In principle, repression systems based on the action of a single factor have an inherent risk of incomplete repression. With inducible promoters, it is hardly possible to investigate all conceivable conditions that, despite good repression, can still lead to unwanted induction. Tissue-specific promoters are similarly limited, since a clear and complete repression in non-target tissues cannot be guaranteed completely. The same applies to the functional inhibition of enzyme activities by fusion with a ligand binding domain.

A conditional functionality of recombinase-mediated DNA elimination from transgenic plants has already been shown and sequence-specific recombinases and their recombination integrated at certain locations in the plant genome are also suitable for the integration of DNA sequences at certain positions in the plant genome. To date, however, there is no known system for controlled sequence equivalence before DNA integration or DNA elimination that would be ready for use. Rather, the disadvantages and limitations described above have also been confirmed in transgenic plants. In addition, in comparison to other eukaryotic organisms for the regulation of a Rec-LBD system, the multitude of secondary ingredients is aggravated by plants. In particular, a cross reaction with naturally occurring steroid-related substances, such as brassino steroids or the like, must be observed. Successful use of the Rec-LBD system in one plant species does not guarantee transferability to another plant species.

So far, sequence-specific recombinases have been used in transgenic plants for the development of marker gene removal systems. Promoters that were inducible by chemicals were used here in particular. However, various embodiments of tetracycline-repressed or -induced promoters were found to be insufficiently suitable for reliably suppressing uncontrolled premature activation of the recombinase. However, the presence of the selectable marker gene is absolutely necessary for the selection of homogeneous, transgenic plants from the Agrobacterium-mediated transformation over a long period of time, since otherwise chimeric plants are formed, some of which are from transgenic cells and some are of non-transgenic mature ones Cells exist. If it is a vegetatively propagated plant species, this leads to an uncontrollability of the plant material used, which is unacceptable from a regulatory point of view alone, but even more from a practical point of view. In the case of a sexually propagated plant species, non-transgenic cells can be sorted out via the pollen or the progeny produced by fertilization and broken down in accordance with Mendel's rules. However, this leads to a considerable Chen reduction in technical efficiency in the development of transgenic plant lines solely for marker gene removal.

In the production of critical proteins, e.g. it is equally important for the plant to be physiologically disadvantageous or even toxic or not to be expressed in the field during the growth phase due to medical activity, so it is also important to be able to exercise extremely reliable control over recombinant activation. A recombinase-induced "gene switch system" can be used for "molecular farming", as has been described in principle with a tissue-specific regulated promoter. If the repression system were not controllable, the containment would not be sufficiently reliable, e.g. for the molecular farming of therapeutic proteins.

Methods have been described to optimize the use of Rec-LBD systems, for example a bidirectional constitutive promoter was used to control the gene to be activated. A selectable marker gene was cloned between this promoter and the gene, flanked by recombination sequences. The Rec-LBD system was cloned behind the free second promoter position. After induction of Rec-LBD by adding the ligand, the marker gene sequence was eliminated, the gene to be activated coming under the control of the constitutive promoter. For use in plants, however, the restriction to a single control system is problematic here as well, and reading through from the constitutive promoter through the marker gene into the gene to be activated can also take place. Furthermore, only very few bidirectional promoters that are active in plants are known, so that there is a strongly limiting factor for practical use. A more complex system has also been described, with the timing still being controlled solely by the addition of ligands. The Rec-LBD gene was cloned under the control of a tissue-specific promoter. In that As a result, tissue is always present with a Rec-LBD protein that has been shown to have residual activities. Although many plant-specific tissue-specific promoters are known, some of which also have a good limitation to the desired tissue, there is a considerable risk due to position effects that there may be large deviations in individual transgenic plant lines from the originally characterized tissue-specific expression profile. In particular, the transferability from one plant species to another is very limited. The tissue-specific regulation can therefore only be used to a limited extent for use in plants.

Thus, the invention is essentially based on the technical problem of providing an elimination system based on the Rec system for a desired DNA sequence which does not have the disadvantages of the methods described in the prior art, i.e. Above all, it ensures that the suppression of recombinase activity, as long as desired, is complete and can be achieved in a desired host organism.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

It has been found in the experiments leading to the present invention that by double-repressing control of the elimination of DNA sequences between recombination DNA sequences in the plant genome (including the plastid and mitochondrial genome) the solution to the above technical problem is achieved can. For this purpose, the repression of the transcription of the recombinase gene via an inducible promoter and the repression of the enzyme function via the Rec-LBD system were combined. By selecting a tissue-specific and inducible promoter at the same time, for which there are now lower quality requirements, it is possible to add an additional tissue. integrate specificity. The double repression has a direct effect on the recombination activity. For example, the transcription of the gene coding for the recombinase is repressed by an inducible promoter which, for example, only becomes active under anaerobic conditions. The gene also codes for a recombinase-LBD fusion protein which is inactivated as long as no ligand is present. Residual transcription and the residual activity of the Rec-LBD fusion protein in transgenic plants are minimized by the synergistic repression effect.

Thus, the present invention relates to a method for the controlled elimination of a desired DNA sequence in a host organism, preferably a transgenic plant, which is characterized in that in step (I) the host organism with (a) those to be eliminated later, 5 'and 3 'DNA sequence flanked by recombination DNA sequences and (b) a ligand-binding-domain-recombinase-fusion protein (Rec-LBD) coding gene which recognizes these recombination DNA sequences and which is under the control of an inducible promoter is transformed under conditions, among which the inducible promoter is repressed, the ligand specifically binding to Rec-LBD being absent; and in step (II) the DNA sequence to be eliminated is eliminated via the expression of Rec-LBD by activating the inducible promoter and the activation of Rec-LBD by adding the ligand which specifically binds to Rec-LBD at the desired point in time.

Methods for constructing the constructs required to carry out the method according to the invention are known to the person skilled in the art and are also described in standard works (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY). The (a) DNA sequence to be eliminated, 5 'and 3' flanked by recombination DNA sequences, and (b) the ligand recognizing the recombination DNA sequences Binding domain recombinase fusion protein (Rec-LBD) encoding DNA sequence under the control of an inducible promoter are both preferably inserted on a vector, the vector preferably being a plasmid, a cosmid, a virus, a bacteriophage or another vector common in genetic engineering. These vectors can have further functional units which bring about a stabilization of the vectors in the host organism, such as a bacterial origin of replication or the 2-micron DNA for stabilization in Saccharomyces cerevisiae. Furthermore, "left border" and "right border" sequences of agrobacterial T-DNA can be contained, which enables a stable integration into the genome of plants. Furthermore, a termination sequence can be present which serves to correctly terminate the transcription and to add a poly-A sequence to the transcript. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be interchanged as desired.

The two DNA sequences (a) and (b) described above can also be inserted on different vectors and the host organism, preferably the transgenic plant, can be transformed simultaneously with both vectors or first with one vector and later with the other vector become. For example, the DNA sequence (a) or (b) can already be integrated into the genome of the host organism and only then can the transformation be carried out with a vector containing the corresponding other DNA sequence. In particular it is mentioned that the recombinase gene is based on a virus, e.g. TMV, can lie and is only activated after infection of the host organism. The DNA sequence (b) can also be part of the DNA sequence (a), i.e. be inserted between the recombination DNA sequences, so that after activation the gene coding for the recombinase itself is excised.

After expression (via promoter activation) and activa tion ^¬ (by adding the binding to Rec-LBD ligands) of the recombinase is carried out on the two recombinant DNA sequences a site-specific recombination and thus an excision of the DNA sequence to be eliminated, whereby, for example, a gene to be expressed is located directly downstream of its promoter, which initiates the transcription and translation and thus the foreign protein biosynthesis. In principle, any recombinase system is suitable for the method according to the invention, whereby according to standard methods the person skilled in the art modifies the gene coding for the recombinase in such a way that it codes for the recombinase as a fusion protein with a ligand-binding protein or a part thereof such that the recombinase is only enzymatically active after binding of the ligand, ie the recombinase is inactive due to the fusion with the LBD in the absence of the ligand. The term "ligand binding domain" or "LBD" as used herein encompasses any protein or protein fragment which, after fusion to the recombinase, is capable of inhibiting recombinase activity in the absence of the ligand and restoring it after adding the ligand. The recombinase-LBD system described in WO 95/00555, in which the ligand activating the recombinase via the LBD is estradiol, is preferred for the process according to the invention. The recombinase systems encompassed by the present invention include not only the systems already described above or in the literature, or systems modified by fusion with an LBD domain, but also systems which differ from the original systems by further modifications, as long as these do not significantly impair the use according to the invention.

A large number of cloning vectors which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells are available for preparing the introduction of a foreign gene into the host organism, for example into higher plants. Examples of such vectors are pBR322, pUC series, Ml3mp series, pACYC184, etc. The foreign gene can be introduced into the vector at a suitable restriction site. The plasmid obtained is used for the transformation of E. coli cells. Transformed E.coli cells are in a suitable medium grown, then harvested and lysed to give the plasmid. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned in the same or different plasmids.

For the introduction of DNA into host organisms, e.g. in a plant host cell, a variety of techniques are available. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the bio-listical method and other possibilities ,

When injecting and electroporation of DNA into plant cells, there are no special requirements for the plasmids used. Simple plasmids, e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, a selectable marker should be present. Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. E.g. If the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, must be connected to the genes to be introduced.

If agrobacteria are used for the transformation, it is advantageous to clone the DNA to be introduced into special plasmids, in particular into an intermediate or a binary vector. The intermediate vectors can be due to sequences that are homologous to sequences in the T-DNA homologous recombination can be integrated into the Ti or Ri plasmid of the agrobacteria. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens using a helper plasmid. Binary vectors can replicate in both E. coli and agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.

For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material, e.g. Leaf pieces, stem segments, roots, protoplasts or suspension-cultivated plant cells can then be used to regenerate whole plants in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. The plants thus obtained can then be examined for the presence of the introduced DNA. Other ways of introducing foreign DNA using the biolistic method or by protoplast fusion are known.

Alternative systems for the transformation of monocotyledonous plants are the transformation using the biolistic approach, the electrically or chemically induced DNA uptake in protoplasts, the electroporation of partially permeabilized cells, the macroinction of DNA in inflorescences, the microinjection of DNA into microspores and Pro-embryos, DNA uptake by germinating pollen and DNA uptake in embryos by swelling (for an overview: Potrykus, Physiol. Plant (1990), 269-273). While the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens has been established, recent work indicates that monocotyledonous plants can also be transformed using Agrobacterium-based vectors.

The transgenic plants useful in the process according to the invention can in principle be plants of any plant species, i.e. both monocot and dicot plants. They are preferably useful plants, in particular plants such as wheat, barley, rice, maize, sugar beet, sugar cane, potatoes, brassicaceae, legumes or tobacco. It can also be algae, mosses and fungi. The parts of plants desired for the elimination of the corresponding DNA sequence in principle relate to any part of the plant, in any case propagation material and harvest products of these plants, e.g. Fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.

In a preferred embodiment of the method according to the invention, the DNA sequence to be eliminated lies between a promoter and a gene and prevents the transcription and / or translation of this gene. Only after expression and activation of the recombinase is there a site-specific recombination between the two recombination DNA sequences and thus an excision of the DNA sequence to be eliminated, as a result of which the desired gene is located directly downstream of the promoter, which means the transcription and translation and thus the foreign -Protein biosynthesis initiated. This procedure allows, for example, use in the post-harvest production of a gene which is under the control of a very strong promoter and which codes for the desired protein to be produced.

The method according to the invention, which, as described above, is characterized by a very dense repression system with regard to the recombinase, can thus also be used for the recombinant production of highly phytotoxic proteins or peptides. tides are used, or for the production of proteins or peptides that have a critical medical effect. A protein or peptide that is toxic to the cell includes, for example, a membrane-disrupting protein or peptide, such as mellittin, magainin, cecropin, attacin, lysozyme, etc., a peptide antibiotic, such as vancomycin, valinomycin, etc., or an RNAse, in particular an RNAse without DNAse activity. In addition, a cytosine deaminase, diphtheria toxin A, herpes simplex virus thymidine kinase type I, a rol protein from Agrobacterium rhizogenes, an antibody against abscisic acid, etc. belong to a protein or peptide that physiologically hinders the growth of the plant.

Furthermore, the method according to the invention can also be used to eliminate marker genes which were originally used for the selection of transformants and which are no longer desired in the transgenic plant at a later point in time. Thus, in a further preferred embodiment of the method according to the invention, the DNA sequence to be eliminated is a selectable marker gene. In an even more preferred embodiment of the method according to the invention, the selectable marker gene encodes an antibiotic-inactivating protein, e.g. Neomycin phosphotransferase II, or it is a gene coding for the "green fluorescent protein".

Furthermore, the method according to the invention can also be used to selectively integrate a second desired DNA sequence into the genome of the host organism after eliminating the desired DNA sequence, for example the selectable marker gene, ie instead of the eliminated DNA sequence between the 5 ' and 3 'recombination DNA sequences are introduced. For this purpose, the host organism can be transformed with a vector containing the second desired DNA sequence, so that the latter DNA sequence can specifically recombine into the genome of the host organism. With regard to the vectors, DNA sequences and conditions suitable for this, reference is made to the above statements. Those skilled in the art are familiar with inducible promoters suitable for the process according to the invention and the inducers (or inhibitors) (gaseous, liquid or solid, volatile compounds) which are suitable for this purpose. Suitable gaseous inductors and conditions for the most efficient exchange of the gas phase are also known to the person skilled in the art. Reference is made to the anaerocult system (Merck, Darmstadt, Germany), which creates an anaerobic environment in which oxygen is bound and C0 _{2 is} released. In this system, the GapC4 promoter from maize is induced anaerobically by the CO ₂ atmosphere (Bülow, L. et al., Molecular Plant-Microbe Interactions (1999), 182-188). The same effect is also achieved by introducing technical nitrogen. Another example is the induction of "pathogenesis related protein" promoters such as L-phenylalanine, ammonium lyase, chalcon synthase or "hydroxyproline rieh glycoprotein" promoters by ethylene (Ecker, JR and Davis, RW, Proc. Natl Acad. Sci. USA 84: 5202-5206 (1987).

The inducible promoters suitable for the process according to the invention also include promoters which can be induced by nebulizing an inductor. Soluble inductors suitable for atomization and conditions for the most efficient atomization are also known to the person skilled in the art. Reference is made to a chimeric transcription induction system which is induced by the soluble inducer dexamethasone (Plant J. 11 (1997) 605-612; Kunkel et al., Nature Biotechnol. 17 (1999), 916-918 ). The corn incwl promoter is activated by the addition of sucrose or D-glucose (Chen, WH et al., Proc. Natl. Acad. Sci. USA 96 (1999), 10512-10517). Many "pathogenesis related protein" promoters are activated by salicylic acid (Gaffney et al., Science 261 (1993), 754-756. A volatile inducer is methyl salicylate, which is converted to salicylate in the receiving plant, which has an inductive effect (Shulaev, V. et al., Nature 385 (1997), 718-721) The nebulization of solutions, for example of promoter-inducing (bio) chemicals, offers the advantage of technically simple and uniform distribution of the inducing substance around and into the tissue to be induced. A rapid adjustment of the uniform distribution is preferably promoted by active circulation of the gas phase. In addition, a simple and efficient compensation of the resulting differences in concentration is achieved, so that controlled process conditions can be guaranteed. For example, the agrochemical RH5992 (Tebufenozide, Rohm & Haas, Croyden, UK) can be used for the nebulization, RH5992 acting as an inducer for the promoter to be induced via a chimeric transcription activator protein (Gatz and Lenk, Trends in Plant Science 3 (1998) , 352-358). The promoter is specifically turned on by RH5992 and is inactive without the presence of this compound. The inductor mist is flushed evenly towards the tissue to be induced, for example by a continuous air distribution. The effective induction of the promoter is achieved by diffusion from the tissue surface into the cells.

Volatile inductors suitable for overflow and promoters inducible by these are likewise known to the person skilled in the art. Particularly worth mentioning is methyl salicylate, which is converted into salicylate in the receiving plant, which, as described above, has an inductive effect (Shulaev, V. et al., Supra). Another example of a volatile inducer is ethanol, which induces the alcA promoter from Aspergillus nidulans in transgenic tobacco (Caddick, M.X. et al., Nature Biotechnology 16 (1998), 177-180). For an active overflow, the procedure is such that the liquid or solid (volatile) inductor is overflowed with a suitable gaseous carrier medium for conversion into the volatile, gaseous phase, and preferably an active circulation of the gas phase is carried out in order to achieve the uniform distribution of the inductor ,

Suitable physically inducible promoters, for example by thermal changes such as heat or cold shock, are also known to the person skilled in the art. To heat inducible promoters include, for example, HSP81-1 promoter from Arabidopsis thaliana (Yabe et al. Plant Cell Physiol. 35 (1994), 1207-1219) Ha hsp 18.6 G2 promoter from sunflower (Coca et al. Plant Mol. Biol. 31 (1996) 863- 876), HSP18.2 promoter from Arabidapsis thaliana in transgenic tobacco (Yoshida et al. Appl. Microbiol. Biotechnol. 44 (1995), 466-472), while the C17 promoter from potato (Kirsch et al. Plant Mol Biol. 33 (1997), 897-909) counts.

Thus, the inducible promoter suitable for the method according to the invention is preferably an anaerobic condition, a chemical or a physically inducible promoter, such as the GapC4 promoter or the Adhl promoter, and the induction takes place via a change in the gas phase in this way that it is an oxygen deprivation; see also the explanations above. The anaerobically inducible GapC4 promoter (DE 195 47 272), e.g. in connection with harvested transgenic plant tissue, e.g. transgenic potato tubers. Most plant promoters are switched off under anaerobic conditions, while the GapC4 promoter is switched off under aerobic conditions and is switched on by simple oxygen deprivation (Bülow et al., Supra). This will e.g. achieved by introducing nitrogen or carbon dioxide into the reaction or storage space. A completely anaerobic environment is thus achieved within a few hours even in an intact potato tuber, as a result of which the promoter induction and Fre d protein expression take place.

In a further preferred embodiment of the method according to the invention, the DNA sequence (b) which codes for the ligand binding domain recombinase fusion protein (Rec-LBD) and is under the control of an inducible promoter is not stably integrated into the host genome, but becomes the desired one time introduced by a systemically ver ^¬ wide forming virus, for example, in the case of plants, based on TMV TMV or vectors. This approach is particularly suitable for the post-harvest production of a desired protein.

The Rec-LBD gene can also be induced in the core of the host, e.g. of the transgenic plant cell. The Rec-LBD protein is fused with an N-terminal transit peptide (responsible for plastid or mitochondrial transport). The protein translated after induction is consequently transported into the plastids or mitochondria. The recombination sequences together with the DNA sequence to be eliminated later are introduced into the plastid or mitochondrial genome by direct plastid or mitochondrial transformation (for example Svab et al., Proc. Natl. Acad. Sci. USA 87) (1990), 8526-8530; Carrer et al., Mol. Gen. Genet. 241 (1993), 49-56; Sidorov et al., Plant J. 19 (1999), 209-216). After promoter induction and addition of the ligand, a recombination takes place in the transformed plastids or mitochondria and the DNA sequence located between the recombination sequences is eliminated. This method is suitable e.g. for the production of proteins with medicinal effects, e.g. on the heart, circulation, organ functions, with angiogenesis induction or with hormone effects. Foreign substances such as polyhydroxyalkanoates, polyhydroxybutyrates or somatotropin can also be prepared (Poirier, Current Opinion in Biotechnology 10, (1999), 181-185; Nawrath et al., Proc.Natl.Aca. Sei. USA 91, (1994), 12760 -12764; Staub et al., Nature Biotechnology 18, (2000), 333-338). The above method is particularly suitable for post-harvest production in transformed plastids or mitochondria. For example, a transcription or translation stop DNA sequence flanked by the two recombination DNA sequences is integrated between the promoter active in plastids or mitochondria and the gene to be expressed and the entire construct is integrated into the plastids or Mitochondria transformed.

Thus, in a further embodiment of the method according to the invention, the ligand binding domain recombinase fusion protein (Rec-LBD) is N-terminal with a transit peptide fused for plastid or mitochondrial delivery and the coding DNA sequence integrated into the nuclear genome, and the gene, whose expression is to be controlled via Rec-LBD, integrated into the plastid or mitochondrial genome, whereby between the promoter for this gene and the gene even a transcription stop sequence flanked by two recombination DNA sequences is incorporated. Examples of these DNA encoding transit peptides are the DNA for the transit peptide of the small subunit of ribulose bisphosphate carboxylase (for plastid localization) (Anderson and Smith, Biochemical Journal 2_40 (1986), 709-715), the DNA for the Flb Subunit of ATP synthase from Nicotiana plumbaginifolia fused with maize T-urf 13 protein (Chaumont et al., Proc. Atl.Acad. Sci. USA 92 (1995), 1167-1171) and the DNA for the mitochondrial tryptophanyl-tRNA- Yeast synthetase fused with GUS (Schmitz and Londsdale, Plant Cell 1 (1989), 783-791).

The present invention also relates to a vector which comprises (a) a DNA sequence to be eliminated, 5 'and 3' flanked by recombination DNA sequences, and (b) a ligand-binding domain-recombinase fusion protein which recognizes these recombination DNA sequences (Rec-LBD) encoding DNA sequence under the control of an inducible promoter as described above. With regard to suitable vectors, we refer to the above explanations.

Finally, the present invention also relates to a host organism which was transformed according to step (I) of the method according to the invention or which contains the vector according to the invention. The host organism is preferably a host organism which was also treated in accordance with step (II) of the process according to the invention. The host organism is preferably a transgenic plant, the term "transgenic plant" also comprising individual parts of plants or plant organs. This also includes seeds, fruits, tubers, rhizomes, seedlings and cuttings. The following transgenic plants are particularly preferred: wheat, barley, maize, rice, sugar beet, sugar cane, potato, brassicaceae, legumes, tobacco, moss, algae and fungi.

The following examples illustrate the invention.

Example 1: Inducibly recombinant controlled post-harvest production of an sc-Fv antibody under the control of a strong promoter

The artsaenko et al. (Molecular Breeding 4 (1998), 313-319) described cDNA, which codes for a scFv (ox) antibody localized in the endoplasmic reticulum with KDΞL-ER targeting sequence, was by means of a linker ligation, on 5 'end with CAT GCC ATG GCA TG [5' phosphorylated oligonucleotide], at the 3 'end with GCT CTA GAG C [5' phosphorylated oligonucleotide]) modified such that they have an Ncol restriction site at the 5 'end and at 3 'end had an Xbal restriction site. After digesting the plasmid pRTlOO (Töpfer et al., Nucleic Acids Research 15 (1987), 5890) with Ncol and Xbal, the scFv-coding DNA fragment was inserted into this expression vector. The plasmid pRTl00 / scFv (ox) E was obtained.

A synthetic nucleic acid was inserted into the Ncol restriction site of pRT100 / scFv (ox), which contained two FRT recombination sequences (Buchholz et al., Nucleic Acids Research 21 (1996), 3118-3119). The plasmid pRTl00 / FRT-scFv (ox) was obtained.

The anaerobically inducible GapC4 promoter from DE 195 47 272 was modified by means of a PCR reaction in such a way that it received a HindIII restriction site at the 5 'end and an Ncol restriction site at the 3' end. The following primer pair was used for the PCR reaction used:

HincII-pGapC4 Primer: 5 '-CAT GTC AAC ACA TAA GGA AGA AGA

GGT AGA AAG-3 ' pGapC4-Ncol Primer: 5 '-CAT GCC ATG GAT CGA TGA CGG GGT TGG CGA GTG TG-3'

The CaMV 35S promoter was removed from the plasmid pRTlOO by means of restriction digestion with HindIII and Ncol. Instead, the nucleic acid fragment described above was ligated in with the GapC4 promoter. The plasmid pRT10OGap was obtained. The cDNA coding for the FLP recombinase-LBD fusion protein (WO 95/00555) was cloned as a PCR-adapted Ncol-Xbal fragment into the Ncol site of pRT10OGap in the sense orientation. The following primer pair was used for the PCR reaction:

NcoI-FLP-LBD Primer: 5 '-CAT GCC ATG CCA CAA TTT GAT ATA TTA TGT AAA AC-3'

FLP-LBD-Xbal Primer: 5'-GCT CTA GAT CAG ACT GTG GCA GGG AAA CCC TC-3 '

The plasmid pRT10OGap / FLP was obtained. After cleavage with HindIII, the expression cassette for the Rec-LBD protein was isolated. After filling in, this HindIII fragment was inserted "blunt end" in anti-orientation with respect to the CaMV 35S promoter and the scFv (ox) gene between the two FRT recombination sequences of the plasmid pRT100 / FRT-scFv (ox). The plasmid pRTl00 / FLP-Gap / scFv (ox) was obtained. From this the entire construct was isolated by restriction digestion with HindIII and inserted into the binary vector pSR 8-30 (Düring et al., Plant Journal 3 (1993), 587-598; Porsch et al., Plant Molecular Biology 37 (1998) , 581-585). The expression vector pSR 8-30 / FLP-Gap / scFv (ox) was obtained, the structure of which is shown schematically in FIG. 1A.

The expression vector pSP 8-30 / FLP-Gap / scFv (ox) was used to transform E. coli S17-1. Transformants were mixed with Agrobacterium GV 3101 and incubated at 28 ° C overnight. (Koncz and Schell, Molecular and General Genetics 204 (1986), 383-396; Koncz. Et al., Proc. Natl. Acad. Sci. USA 94 (1987), 131-135). It was carbenicillin- lesson, the bla gene required for this was present in the above expression vectors. Selection clones of Agrobacterium tumefaciens were cut from leaves of the potato plant cv and cut several times on the middle rib. Desiree applied and the leaves were incubated for 2 days at 20 ° C in the dark. The agrobacteria were then washed off and plant growth substances were added to the potato leaves, so that shoots regenerated preferentially. Furthermore, by adding kanamycin to the plant medium, non-transformed cells in the potato leaves were killed. Growing shoots were cut off and rooted on the medium without plant growth substances, but with kanamycin (100 mg / l). The further cultivation of the potato plants was carried out in the usual way. To induce the recombinase, cut leaf material or intact or cut tuber material was carried out using the Anerocult system (Merck, Darmstadt, Germany) as described in Bülow et al. (Molecular Plant-Microbe Interactions 12 (1999), 182-188). After 40 hours, the plant material was sprayed with a solution of 10 ^-6 M estradiol and cultivated for a further 2 days. The plant material was then mortared. The detection of the expressed scFv antibody was carried out via the c-myc "tag" contained using the monoclonal antibody 9E10-IgG (Cambridge Research Chemicals, Northwich, Cheshire, UK) or Protein L (Clontech, Palo Alto, CA., USA ) in a Western blot or ELISA. For this purpose, the total protein of the potato material was isolated and used in the corresponding detection methods. Transgenic potato plants found to be expression-positive were grown in the greenhouse in a pot or in a straw bed or in the open under normal horticultural or agricultural conditions. The tubers were harvested and stored after normal handling.

For the post-harvest production of the scFv antibody, the tubers were placed in a reaction container made of steel or plastic, which has a gas supply valve at the bottom and a gas discharge valve at the top. The room air in the tank became quick displaced by the supply of technical nitrogen or carbon dioxide. A constant composition of the gas phase in the reaction vessel was set under slow air flow (1 m ³ gas supply per hour per m ² base area). After 40 hours, the plant material was sprayed with a solution of 10 ^"6 M eastern Radiol and cultured for further 2 days. The tubers were then removed from the reaction container, homogenized, the solids content was centrifuged off and the aqueous supernatant of the chromatographic purification of the scFv In contrast to the induced, no scFv (ox) could be detected in non-induced leaf or tuber tissue.

Example 2: Inducible marker gene removal by using the method according to the invention

The HindIII restriction site located upstream of the GapC4 promoter was opened by partial digestion of the plasmid pRT10OGap. A synthetic nucleic acid was inserted into this interface, which contained an FRT recombination sequence (Buchholz et al., Supra) and which reconstituted the HindIII interface at the 5 'end. With a further partial HindIII digestion, the HindIII interface located downstream of the terminator was opened and a further synthetic nucleic acid was inserted which contained an FRT recombination sequence and which reconstituted the HindIII interface at the 3 ′ end. The plasmid pRTlOOGapFRT was obtained.

The cDNA coding for the FLP recombinase-LBD fusion protein (WO 95/00555) was cloned as a PCR-adapted Ncol-Xbal fragment into the Ncol site of pRTlOOGapFRT in the sense orientation. The following primer pair was used for the PCR reaction:

NcoI-FLP-LBD Primer: 5 '-CAT GCC ATG CCA CAA TTT GAT ATA

TTA TGT AAA AC-3 'FLP-LBD-Xbal Primer: 5' -GCT CTA GAT CAG ACT GTG GCA GGG

AAA CCC TC-3 ' The plasmid pRTlOOGapFRT / FLP was obtained. The expression cassette for the selectable nptII marker gene, containing the CaMV 35S promoter and terminator (Odell et al., Nature 313 (1995), was converted from the plasmid pLH9000 (Hausmann and Töpfer, lectures Plant Breeding 45 (1999), 155-172). 810-812) and in between the neomycin phosphotransferase II gene (nptll) (Beck et al., Gene 19 (1982), 327-336) as Xbal / Sfil fragment by restriction digestion with Xbal and Sfil. This expression cassette was inserted in the sense orientation into the opened Xbal site of the plasmid pRTlOOGapFRT / FLP by filling in overhanging ends after the first "sticky end" ligation step and then performing a "blunt end" ligation as the second step. The plasmid pRTlOOGapFRT / FLP-NPT was obtained. From this, the entire construct was isolated by restriction digestion with HindIII and inserted into the binary vector pSR 8-30. The expression vector pSR 8-30 / Gap-FLP-NPT was obtained, the structure of which is shown schematically in FIG. 1B.

The potato transformation was carried out as described in Example 1. The anaerobic induction of the transcription of the recombinase gene was also carried out in cut sheet material on MS medium analogously to that described in Example 1. The activation of the FLP-LBD recombinase was carried out simultaneously by addition of 10 ^"6 M estradiol in the medium. Induction was carried out over a period of one week. After the induction, the sheet material was cut into small pieces and designed to shoot induction medium. Regenerated shoots were without selection 100 shoots were regenerated, which were analyzed by Southern hybridization with the nptll gene probe for elimination of the nptll gene.

nptll gene probe: 5 '-ACAACAGACAATCGGCTGC

3 '-TCAAGAAGGCGATAGAAGGC

It was found that the nptll gene was no longer present in a large number of the regenerated shoots.

Claims

claims

1. A method for the controlled elimination of a desired DNA sequence in a host organism, characterized in that in step (I) the host organism with

(a) the DNA sequence to be eliminated later, 5 'and 3' flanked by recombination DNA sequences and

(b) a ligand-binding-domain-recombinase-fusion protein (Rec-LBD) encoding these recombinant DNA sequences, which is under the control of an inducible promoter, is transformed under conditions under which the inducible promoter is repressed, the specific being Rec-LBD binding ligand is absent; and in step (II) the DNA sequence to be eliminated is eliminated via the expression of Rec-LBD by activating the inducible promoter and the activation of Rec-LBD by adding the ligand which specifically binds to Rec-LBD at the desired point in time.

2. The method according to claim 1, wherein the DNA sequence to be eliminated lies between a promoter and a gene and prevents the transcription and / or translation of the gene.

3. The method of claim 2, wherein the gene encodes a "cell toxic protein or peptide.

4. The method of claim 3, wherein the cell-toxic protein or peptide is an RNAse.

5. The method of claim 3, wherein the cell-toxic protein or peptide is a membrane-disrupting protein or peptide.

6. The method according to claim 5, wherein the membrane-disrupting protein or peptide is mellittin, magainin, cecropin, attacine or lysozyme.

7. The method according to claim 2, wherein the gene codes for a growth of the host organism physiologically inhibiting protein or peptide.

8. The method according to claim 1, wherein the DNA sequence to be eliminated is a selectable marker gene.

9. The method of claim 8, ^'wherein the selectable marker gene for an antibiotic inactivating protein.

10. The method according to claim 8, wherein the selectable marker gene codes for a "green fluorescent protein".

11. The method according to any one of claims 1 to 10, wherein the inducible promoter is an anaerobic, by a chemical or physically inducible promoter.

12. The method of claim 11, wherein the anaerobically inducible promoter is the GapC4 promoter.

13. The method according to any one of claims 1 to 12, wherein the ligand-binding domain-recombinase-fusion protein (Rec-LBD) coding the recombinant DNA sequences, under the control of an inducible promoter

^" standing DNA sequence (b) is not stably integrated into the host genome, but is only introduced at the desired time by a systemically spreading virus.

14. The method according to any one of claims 1 to 13, wherein the ligand binding domain recombinase fusion protein (Rec-LBD) is N-terminally fused with a transit peptide for plastid or mitochondrial transport, which is therefor coding DNA sequence is integrated into the nuclear genome and the gene whose expression is to be controlled via Rec-LBD is integrated into the plastid or mitoehondrial genome, one of two recombination DNA between the promoter for this gene and the gene itself Sequences flanked transcription stop sequence is built.

15. Vector containing

(a) a DNA sequence to be eliminated, 5 'and 3' flanked by recombination DNA sequences and

(b) a ligand-binding-domain-recombinase-fusion protein (Rec-LBD) coding for these recombination DNA sequences and coding and under the control of an inducible promoter as defined in any one of claims 1 to 14.

16. Host organism which was transformed according to step (I) of the method according to one of claims 1 to 14 or contains the vector according to claim 15.

17. Host organism according to claim 16, which was also treated according to step (II) of the method according to any one of claims 1 to 15.

18. Host organism according to claim 16 or 17, which is a transgenic plant.

19. The transgenic plant of claim 18, wherein the transgenic ^"plant is wheat, barley, maize, rice, sugar beet, sugar cane, potato, a Brassicacee, a legume, tobacco, a moss, an alga or a fungus.