WO2002061056A2

WO2002061056A2 - Degp2 protease that degrades photo-damaged d1 protein and a dna sequence that codes for said protease

Info

Publication number: WO2002061056A2
Application number: PCT/DE2001/004301
Authority: WO
Inventors: Iwona Adamska; Kirsten HAUßÜHL
Original assignee: Mpb Cologne Gmbh
Priority date: 2001-01-31
Filing date: 2001-11-14
Publication date: 2002-08-08
Also published as: DE10104595A1; AU2002216919A1; WO2002061056A3

Abstract

The invention relates to the DegP2 protease, which carries out the primary cleavage of the photo-damaged D1 protein in plants and to DNA sequences, which code for DegP2. The invention also relates to antibodies that bind to DegP2 and to antisense RNAs or ribozymes that are directed against the expression of DegP2. The invention further relates to the use of DegP2 or DNA sequences that code therefor for protecting plants against light-induced damage.

Description

DegP2 protease and this DNA sequence encoding photo-damaged Dl protein

The present invention relates to the protease DegP2, which carries out the primary cleavage of the photo-damaged Dl protein in plants, and to proteins related to DegP2 and DNA sequences coding for these proteins. The present invention further relates to antibodies binding to DegP2 or proteins related thereto, and to antisense RNAs or ribozymes directed against the expression of DegP2. Finally, the present invention relates to the use of DegP2 or the DNA sequences encoding it for protecting plants against damage induced by light.

Organisms carrying out oxygen photosynthesis experience an inhibition of their photosynthetic function (photoinhibition) when exposed to excessive lighting. At normal temperatures, photoinhibition primarily occurs at the level of Photosystem II (PSII), in which a reversible inactivation of PSII is involved due to an arrest of the electron transport within this complex and afterwards an irreversible damage to the subunits of the PSII reaction center. In higher plants, algae and cyanobacteria, PSII is a complex consisting of several subunits and comprising more than 25 different protein subunits. At the core of the complex is the reaction center, which consists of two related proteins Dl and D2, which bind all the cofactors involved in primary and secondary electron transport. Within the PSII reaction center, the Dl protein is the main target for oxidative damage. The rapid breakdown of the photo-damaged Dl protein and its exchange for a de novo synthesized active copy represent an important repair mechanism which is of crucial importance for the survival of the plant under conditions of light stress. By analogy to the L subunit of the bacterial reaction center, the Dl protein was believed to have five transmembrane helices through stroma and lumen loops are connected. The Dl protein is broken down in two steps. The primary cleavage occurs at the stroma loop, which connects the transmembrane helices D and E, whereby a C-terminal proteolytic 10 kD fragment is formed. The proteases involved were previously unknown. The second step of DI degradation involves further cleavage of the primary degradation product, which is believed to involve the FtsH metalloprotease of the thylakoid membrane. Due to the still insufficient knowledge of the components involved in the repair mechanism described above, in particular with regard to the in vivo exchange of photo-damaged DI protein for a functional copy, there have so far been hardly any possibilities for protecting plants, in particular useful plants, from light damage.

The present invention is therefore based on the technical problem of providing means which allow plants to be protected from light damage or a faster recovery of the plants from light damage which has already occurred.

This technical problem is solved by providing the embodiments characterized in the claims. Surprisingly, a gene was found in Arabidopsis which encodes a chloroplast ho ologes of the prokaryotic serine protease of the trypsin type Deg / Htr, the DegP2 protease. This is peripherally associated with the outer surface of the thylakoid membrane. It could also be shown in the investigations carried out on the present invention that the DegP2 protein level rose in response to a high NaCl concentration, dehydration and illumination with strong light. Finally it was shown that the physiological target of the DegP2 protease is the damaged Dl protein from PSII: The DegP2 protease performs the primary cleavage of the Dl protein on the stroma-DE loop, whereby the typical proteolytic fragments are generated. It can be assumed that by increasing the concentration of DegP2 in the chloroplasts, for example by transforming the plants with the gene coding for DegP2 Protection of plants from light damage or a faster recovery of the plants from light damage that has already occurred can be achieved. Furthermore, it can be assumed that this can also provide better protection against high salt concentrations or dehydration.

The present invention thus relates to a DNA sequence which encodes the protease DegP2 with an amino acid sequence shown in FIG. 1B. This DNA sequence preferably comprises the coding region of the DNA sequence shown in FIG. 1.

The present invention further relates to a DNA sequence encoding a protein with the biological properties of DegP2, i.e. a protein which can perform the primary cleavage of the Dl protein in plants and which differs from the above DNA sequence in the codon sequence due to the degeneracy of the genetic code which hybridizes with the above DNA sequences which lead to the coding region 1A has a homology of at least 65%, preferably 75%, more preferably 85% or 90% and most preferably 95%, or which has a fragment, an allelic variant or another variant of the above DNA Sequences is.

The term "hybridize" used in the present invention refers to conventional hybridization conditions, preferably to hybridization conditions in which 5xSSPE, 1% SDS, lxDenhart ts solution is used and / or the hybridization temperatures between 35 ° C and 70 ° C, preferably 65 ° C. After hybridization, washing is preferably carried out first with 2xSSC, 1% SDS and then with 0.2xSSC at temperatures between 35 ° C and 70 ° C, preferably at 65 ° C (for the definition of SSPE, SSC and Denhardts solution see Sambrook et al ., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Stringent hybridization conditions are particularly preferred, as described, for example, in Sambrook et al. , supra.

The terms "variant" or "fragment" used in the present invention encompass DNA sequences which differ from the sequences indicated in FIG. 1 by deletion (s), insertion (s), exchange (s) and / or others in the prior art Differentiate between known modifications or comprise a fragment of the original DNA sequence, the protein encoded by these DNA sequences still having the biological properties of DegP2 and being biologically active in plants. This also includes allele variants. Methods for generating the above changes in the DNA sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al. , supra. Those skilled in the art will also be able to determine whether a protein encoded by such a nucleic acid sequence still has the biological properties of DegP2, e.g. using the assays illustrated in the examples below.

The DNA sequences of the invention are useful for various purposes. Thus, as described in more detail below, they can be used for the recombinant production of the DegP2 protein for analysis purposes, for further characterization or also for protecting plants from light damage or for achieving a faster recovery of the plants from light damage which has already occurred. They can also serve as hybridization probes for the identification of new, related DNA sequences or as a source of information for the development of PCR primers. Finally, they can be used to generate anti-protein antibodies, for example by means of immunization methods, and as an antigen to produce antibodies.

It is also conceivable that in certain cases or under certain conditions a reduced activity and / or concentration of DegP2 in the cell compartments, for example chloroplasts, is desirable. This can be done, for example, by the Lowering or inhibition of the expression of the DNA sequences according to the invention can be achieved. A further preferred embodiment of the present invention therefore relates to an antisense RNA, which is characterized in that it is complementary to the above DNA sequences and can bind specifically to the mRNA translated therefrom and the synthesis of the DegP2- encoded by these DNA sequences Protease or a related protein can reduce or inhibit, and a ribozyme, which is characterized in that it is complementary to the above DNA sequences and can specifically bind to and cleave the mRNA transcribed from these DNA sequences, thereby the synthesis the DegP2 protease encoded by these DNA sequences is also reduced or inhibited. These antisense RNAs and ribozymes are preferably complementary to a coding region of the mRNA. The person skilled in the art is able to prepare and use suitable antisense RNAs based on the disclosed DNA sequences. Suitable procedures are described, for example, in EB-Bl 0223 399 or EP-Al 0 458. Ribozymes are RNA enzymes and consist of a single strand of RNA. These can intermolecularly cleave other RNAs, for example the mRNAs transcribed by the DNA sequences according to the invention. In principle, these ribozymes must have two domains, (1) a catalytic domain and, (2) a domain that is complementary to the target RNA and can bind to it, which is a prerequisite for cleaving the target RNA. Based on procedures described in the literature, it is now possible to construct specific ribozymes that cut a desired RNA at a specific, pre-selected location (see, for example, Tanner et al., In: Antisense Research and Applications, CRC Press, Inc. (1993 ), 415-426). The above ribozymes or antisense RNAs preferably hybridize over a range of at least 15, more preferably at least 25 and most preferably at least 50 bases with the mRNA transcribed by the DNA sequences according to the invention. A reduced activity and / or concentration of DegP2 in the chloroplasts is desirable, for example, to change photosynthesis. To a certain extent, a repressed amount of DegP2 represents one inducible or constitutive negative selection markers, for example because the leaves should fade and the plants die. Coupled with the amount of DegP2, a change in the ascorbic acid content is also to be expected, which can contribute to an improvement in taste when the plant is consumed. For example, a reduced amount of DegP2 could result in a higher concentration of ascorbic acid in the plant, whereby in some plants (e.g. peas) a higher concentration of ascorbic acid is associated with a better taste.

The DNA sequences according to the invention or the DNAs encoding the antisense RNAs or ribozymes described above can also be inserted into a vector or expression vector. The present invention thus also includes vectors or expression vectors containing these DNA sequences. The term "vector" refers to a plasmid (pUClδ, pBR322, pBlueScript etc.), a virus or another suitable vehicle. In a preferred embodiment, the DNA sequences according to the invention are functionally linked in the vector to regulatory elements which allow their expression in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, for example a promoter, such vectors typically contain an origin of replication and specific genes which allow the phenotypic selection of a transformed host cell. The regulatory elements for expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GALl promoter in yeast and the CMV, SV40, RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells. Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable expression vectors for E. coli include, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. Vectors suitable for expression in yeast include pYlOQ and Ycpadl, pMSXND, pKCR, pEFBOS, cDM8 and pCEV4 for expression in mammalian cells. The expression vectors according to the invention also include Baculovirus-derived vectors for expression in insect cells, for example pAcSGHisNT-A.

General methods known in the art can be used to construct expression vectors containing the DNA sequences of the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination, as described, for example, in Sambrook et al. , supra. 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 exchanged as desired. To achieve the localization of the DegP2 protease in the desired compartment of the plant cell, e.g. Chloroplasts or chromoplasts or any other cell compartment, this is preferably expressed as a protein containing the transit peptide shown in FIG. 2B in an expression vector, whereby this transit peptide can be replaced by another transit peptide for the compartment localization. Suitable alternative transit peptides and DNA sequences coding these are known to the person skilled in the art.

To prepare the introduction of the DNA sequences according to the invention into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, Ml3mp series, pACYCl84, 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 used. Transformed E. coli cells are grown in a suitable medium, then harvested and lysed, whereby the plasmid is obtained. 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 into the same or different plasmids.

For the introduction of DNA, 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 Agrobacteriυm tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic 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, by homologous recombination in the Ti or Ri plasmid of the Agrobak teries are integrated. 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 tu efaciens 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 regenerated again 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. Alternative systems for the transformation of plants are the transformation using the biolistic approach, the electrically or chemically induced DNA uptake in protoplasts, the electroporation of partially permeabilized cells, the macro-injection of DNA into inflorescences, the micro-injection 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 has indicated that monocotyledonous plants are also accessible for transformation by means of vectors based on Agrobacterium.

In principle, the suitable plants can be plants act on any plant species, ie both monocot and dicot plants. They are preferably (useful) plants from the groups Gramineae, Chenopodia, leguminous plants, Brasicaceae, Solanaceae, fungi, mosses and algae, in particular plants such as wheat, barley, rice, maize, sugar beet, sugar cane, potato, rapeseed, mustard , Beets, flax, peas, beans, lupine and tobacco. The parts of the plants desired for the expression of the desired protein or the treatment with the chemical inducers in principle relate to any part of the plant, in any case propagation material and harvest products of these plants, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.

The DNA sequences according to the invention can also be inserted into a vector in connection with a DNA coding for another protein or peptide such that the DNA sequences according to the invention are, for example, in the form of a fusion protein, e.g. can be expressed with a His tag to facilitate purification after recombinant production.

The present invention also relates to host cells containing the vectors described above. These host cells include bacteria (for example the E. coli strains HB101, DH1, xl776, JM101, JM109, BL21 and SG13009), yeast, preferably S. cerevisiae, insect cells, preferably sf9 cells, and animal cells, preferably mammalian cells. Preferred mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293 and WI38 cells. Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the DNA molecules according to the invention using the vectors described above are known in the art.

The present invention further relates to a DegP2 protease encoded by the DNA sequences according to the invention or a protein with their biological activity and to methods for their recombinant production using the expression vectors according to the invention. The method according to the invention comprises the cultivation of those described above Host cells under conditions which allow expression of the protein (or fusion protein) (preferably stable expression) and the extraction of the protein from the culture or from the host cells. The skilled worker is familiar with conditions for culturing transformed or transfected host cells. Suitable purification methods (for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are also generally known. The DegP2 protease according to the invention or the protein related to it is not only useful in the measures described above or below, but can also be used, for example, in biological assays for determining activity, for example in combination with a large number of other proteins for a " high-throughput "-Screenen. In addition, it can be useful, for example, for the production of antibodies, as a reagent (for example in labeled form) in competitive assays for the quantitative determination of the protein in plants, as a marker for parts of plants in which the corresponding protein is preferably produced (either constitutively or during a certain stage in terms of tissue differentiation etc.), and also for the isolation of interacting proteins. Finally, the DegP2 protease according to the invention or the protein related to it can also be used to isolate inhibitors, antagonists or agonists, which may be peptides or other small molecules, for example.

Another preferred embodiment of the present invention relates to ligands, for example antibodies, against the proteins according to the invention described above (DegP2 protease or related proteins). These ligands can be used in diagnostic assays, for example. Methods for the isolation or synthesis of such ligands are known to the person skilled in the art. For example, useful activity-inhibiting compounds may be screened in extracts from natural products as a starting material. Such extracts can originate, for example, from fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. The ligand according to the invention is preferably an antibody or fragments thereof which bind specifically to the DegP2 protease or a protein with its biological activity. These antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this context, the term “fragment” means all parts of the monoclonal antibody (for example Fab, Fv or “single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art. The antibodies according to the invention are preferably monoclonal antibodies. The antibodies according to the invention can be produced according to standard methods, the protein encoded by the DNA sequences according to the invention or a synthetic fragment thereof preferably serving as an immunogen. Methods for obtaining monoclonal antibodies are known to the person skilled in the art. The antibodies according to the invention can also be used, for example, for immunoprecipitation of the DegP2 protease according to the invention, for isolating related proteins from cDNA expression banks, etc. The present invention also relates to a hybridoma that produces the monoclonal antibody described above.

The present invention also relates to the use of the DNA sequences according to the invention, of the expression vector according to the invention or of the DegP2 protease or of the protein with their biological activity for protecting plants from light damage or in order to achieve a faster recovery of the plants from light damage which has already occurred. The present invention also relates to the use of the above-described antisense RNA, the ribozyme or the antibody for lowering or suppressing the breakdown of the Dl protein.

Since the DegP2 protease of the invention has homology to bacterial DegP / HtrA protease (see FIG. 2A), which is believed to be involved in the development of tolerance in plants against various stress factors such as oxidation, salt, pH and heat (Spiess et al., Cell 97 (1999), 339-347), it can be assumed that the DegP2 protease has a comparable activity spectrum. The present invention thus also relates to the use of the DNA sequences according to the invention, the expression vector according to the invention or the DegP2 protease or the protein with their biological activity for protecting plants against oxidation (for example after herbicide treatment), salt, pH or heat -Stress.

Since DegP2 plays an important role in photosynthesis, a manipulation of the amount of DegP2 protein can increase the yield. This increase in yield can affect the amount of plants, i.e. that more plants grow under the same cultivation conditions or may affect the yield of ingredients obtainable per plant.

Finally, the present invention relates to a kit which contains a DNA sequence according to the invention or the antibody described above. Depending on the design of the diagnostic kit, the DNA sequence or the antibody can be immobilized. The antibody can be bound, for example, in liquid phase immunoassays or to a solid support. The antibody can be used in different ways and be marked. Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA.

Brief description of the figures:

Figure 1:

(A) DNA sequence for DeαP2

The coding area and the 5 'UTR and 3'UTR are shown

(B) DNA sequence and deduced amino acid sequence

Figure 2: A single copy of the nuclear gene DegP2 in A. thaliana encodes a presumed homologue of the Deg / Htr protease family of the Svnechocvstis belonging to the Cvonabacteria. PCC6803 strain

(A) Sequence comparison of the conserved regions of the HtrA, HhoA and HhoB proteases from Svnechocvstis sv. , DegPl from Arabidopsis and the new DegP2 protease

The sequences were compared manually using the CLUSTALW program. Identical amino acids are shown on a gray background. Gaps are represented by dashes and catalytic amino acids are marked in light gray by an asterisk.

(B) Schematic representation of the predicted structure of DegP2

(C) Southern blot analysis of genomic DNA cut with restriction enzymes and incubated with a DegP2 probe under low stringency conditions

FIG. 3: DegP2 is an endopeotidease of the serine type (A). An E. coli extract without recombinant DegP2 (control), with overexpressed DegP2 and DegP2 purified via His-Tag was tested on activity gels which contained gelatin as a non-specific protease substrate ,

(B) To classify the DegP2 protease according to its catalytic center, activity gels were absent (Control) or the presence of protease inhibitors which were directed against serine (PMSF, DCI and aprotinin), cysteine (E-64), aspartate (pepstatin) or metalloproteases (EDTA).

Figure 4: DegP2 is an extrinsic thylakoid membrane protein which is located in the stroma side of the stroma lamellae and not closely adjacent regions of the grana stack.

(A) Isolated intact Arajbidopsis chloroplasts were separated into stroma, thylakoid membrane and thylakoid lumen fractions. Proteins from each fraction were separated on SDS-PAGE and DepP2 was localized using immunoblots.

(B) The localization of DegP2 in tight and not tight areas of the thylakoid membranes was examined by immunoblots. As a reference, the distribution of the Dl protein from the PSII reaction center and the A subunit of the PSI reaction center (PSI-A) was visualized under the same experimental conditions.

(C) The topology of the DegP2 protease in the thylakoid membranes was tested by incubating the membranes in the absence (control) or presence of 0.5 M NaCl or 0.5 M CaCl ₂ . The membrane pellet (M) and the extracted protein-containing supernatant (S) were analyzed by SDS-PAGE and immunoblots. As a reference, the distribution of the 33 kD protein from PsbO was tested via SDS-PAGE and Coomassie blue staining.

(D) A protease protection assay was performed after adding trypsin (50 µg / ml) to the isolated thylakoid membranes (1 mg chlorophyll / ml) and incubating the samples at 4 ° (30 min.). The protection of DegP2 against trypsin degradation was investigated using immunoblots. Protection of PsbO, a peripheral protein located on the lumen side of the membrane, is shown for reference.

Figure 5: Expression pattern of DeoP2 under different stress conditions (A) AraJidopsis leaves were exposed to different stress conditions for two hours and the total RNA isolated and used for Northern blot analyzes. The rRNA pattern shown by ethidium bromide is shown as a reference.

(B) Western blot analyzes of the DegP2 level after the leaves were subjected to various stress conditions for two hours. As a reference, the Lhcb2 level was tested via SDS-PAGE and Coomassie blue staining.

Figure 6: DegP2 mediates GTP-dependent the primary cleavage of the damaged Dl protein from the PSII reaction center

(A) Isolated heat-treated thylakoid membranes were incubated in the absence (-DegP2) or presence (+ DegP2) of purified recombinant DegP2 and the degradation of the Dl protein was determined by immunoblotting after 30 min. Incubation at 37 ° C. Immunoblots, which show the α-subunit of Fl ATPase (CFl-α), various subunits of the PSI complex and Lhcb2, confirm the selectivity of the spectrum of action of DegP2.

(B) Isolated heat-treated thylakoid membranes were reconstituted with purified recombinant DegP2 fractions and the degradation of Dl was examined by immunoblotting after incubation of the samples at 4 ° C or 37 ° C for 6 hours. As negative controls, E. coli fractions without expressed DegP2 (control) or fractions with the overexpressed non-proteolytic class la metallothionein (MTla) with thylakoid membranes were reconstituted and analyzed in parallel.

(C) Kinetics of Dl protein degradation in heat-treated thylakoid membranes in the absence (-GTP) or presence (+ GTP) of 2 mM GTP. Thylakoid membranes were incubated either with purified recombinant DegP2 (+ DegP2) or with E. coli fractions containing overexpressed MTla (- DegP2).

(D) Isolated thylakoid membranes were washed with 0.5 M NaCl to remove endogenous DegP2 activity and the Dl protein was photo-damaged by illumination at 5000 μmol / m ² / s (90 min., 0 ° C.). The thylakoid membranes were then used for reconstitution with purified recombinant DegP2 and the degradation of the Dl protein was examined by immunoblots (upper and middle sections). In control assays, the degradation of CP43 and D2 was examined by immunoblots 120 min after incubation with specimens pretreated with high light in the presence or absence of purified recombinant DegP2 (lower section)

Figure 7: Model of the degradation of the DI protein

The DegP2 protease attached peripherally to the stroma side of the thylakoid membranes performs the primary cleavage of the photo-damaged Dl protein of the PSII reaction center at the stroma loop between helices D and E, thereby producing proteolytic fragments with 23 and 10 kD. At the same time, a newly synthesized functional copy replaces the degraded DL protein. The 23 kD fragment is further broken down by an integral membrane metalloendopeptidase.

The following examples illustrate the invention.

Example 1: General procedures

(A) Plant breeding / stress conditions

Arabidopsis thaliana L. cv. Columbia was grown in a growing chamber on earth at 25 ° C and with a light intensity of 100 μmol / m ² / s under short day conditions. The light stress conditions were carried out on adult leaves which had been separated from 4 to 5 week old plants. The leaves were placed on water and exposed to light with an intensity of 2500 μmol / m ² / s. With regard to cold or heat stress, the separated leaves lying on water were transferred to an incubator at 4 or 42 ° C. for 2 hours. Dewatering stress was carried out with light at an intensity of 10 μmol / m ² / s on leaves that had been dehydrated on Whatmann 3MM paper at room temperature. After two hours of incubation, the relative water content of the leaves was reduced to 50%. To achieve high salt stress or oxidative stress, the Leaves immersed for two hours in 400 mM NaCl or in a 2% H ₂ 0 ₂ solution. UV-A treatment was carried out by illuminating the separated leaves with a UV lamp (366 nm; two hours) at a light intensity of 25 μmol / m ² / s. The plant material was collected and either used immediately for extractions or frozen in liquid nitrogen and stored at -70 ° C. for subsequent preparations.

(B) Cloning the DegP2 gene

A primer pair was designed based on chromosome 2 localized ORF F17A22.23, which was thought to encode DegP2 protease in Arabidopsis (5'-AGT GCC GCC TCC GTA GCA-3 'and 5' -TTA TGC CCA CAC CAG TCC ATC-3 ') and for the amplification of DegP2 ORF from the λ-ZAP (Stratagene, Heidelberg). cDNA library (1 x 10 ³ pfu) used, which was made from Arabidopsis seedlings. The PCR cycle profile was: 1 min. Denaturation at 94 ° C, ql min. Annealing at 50 ° C and 2 min. Primer extension at 72 ° C; 30 cycles. The amplified cDNA fragment (1821 bp) was purified (Qiaex; Qiagen, Hilden) and cloned into the cloning vector pCR2.1 (Invitrogen, San Diego) before the sequencing of the two DNA strands (CyberGene, Stockholm, Sweden). A search with the DegP2 sequence obtained in an EST database (TIGR) identified the Arabidopsis EST clone 133N20T7 (2280 bp) as identical to DegP2.

(C) DNA analysis

Genomic DNA was extracted from leaves as described by Doyle and Doyle (Focus 12 (1990), 13-15). The DNA (10 μg per reaction) was digested with restriction enzymes, then electrophoresed on a 0.8% agarose gel and transferred to "Hybond N +" membrane (Amersham Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions. The cDNA probe was labeled with [α- ³² P] CTP using the "Megapri e DNA labeling kit" from Amersham Pharmacia. The hybridization and washing steps became lower and higher under conditions Stringency according to Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)).

(D) RNA analysis

Total RNA was extracted from control or stress-treated leaves using the "RNeasy mini kit" (Qiagen) according to the manufacturer's instructions. After 5 μg of RNA had been separated on a 1.2% agarose gel, it was transferred to a “Hybond N ⁺ ” membrane according to Sambrook (see above) before the hybridization.

(E) Overexpression of DeqP2 in E. coli and production of polyclonal antibodies

The DegP2 cDNA was amplified using the primers 5 '-GCC GCC TCC GTA GCA AAC-3'^' and 5' -TGC CCA CAC CAG TCC ATC A-3 'and the cDNA plasmid of the EST clone 133N20T7 as a template. The DegP2 protein was used in E. coli as a fusion protein with the N-terminally linked thioredoxin and the C-terminally linked His tag (His ₆ ) (using the kit "pBAD / Topo ™ ThioFusion ™ Expression System, Invitrogen). The recombinant DegP2 protein was purified under denaturing conditions using affinity chromatography on Ni-NTA agarose (“Qiaexpress”; Qiagen) and eluted from the column with 200 mM imidazole. The fractions containing DegP2 were analyzed by SDS- PAGE separated and transferred to nitrocellulose membranes before the generation of polyclonal antibodies in rabbits (BioGenes, Berlin, Germany).

(F) Protein analyzes

Isolated thylakoid membranes and stroma proteins were separated using SDS-PAGE, generally using 10% polyacrylamide mini gels (Hoefer mini gel system; Amersham Pharmacia Biotech, Uppsala, Sweden). The gels were loaded with equal amounts of protein and immunoblotting according to Towbin et al. (PNAS USA 76 (1979), 4350-4354) using a "enhanced chemical luminescence" system (ECL; Amersham International, Buckinghamshire, Great Britain) as a detection system.

(G) activity assays and inhibitor studies

For activity assays, 1 μg of purified DegP2 protease (with His-TAG) at 4 ° C. on 10% SDS polyacrylamide gels with 0.2% gelatin (type "B bloom 75"; Sigma, Deisenhofen) as a non-specific protease substrate separated. The gels were then incubated for 30 minutes at 25 ° C. in 50 M Tris (pH 8.0), 5 mM MgCl ₂ and 1% Triton-X? "To exchange SDS and to renaturate the DegP2 protease Buffer was exchanged for a buffer without detergent and the gel was incubated for a further 16 hours at 37 ° C. to allow the gelatin to degrade. After staining the gel with a Coomassie blue solution (40% ethanol, 10% acetic acid, 0.1% Coomassie Blue R-250) and decolorization with a solution of 40% ethanol and 10% acetic acid, a white zone was visible on a blue background which corresponded to the position of the active DegP2 protease.

To classify the DegP2 protease, activity gels in the absence (control) or presence of protease inhibitors (100 μg / ml PMSF, 10 μg / ml DCI, 1 μg / l aprotinin, 1 μg / ml E64 [3-carboxy-trans-2 , 3-epoxypropyl-leucylamido (4-guanidino) butane), 1 μg / ml pepstatin and 500 μg / ml EDTA according to the manufacturer's protocols (Boehringer Mannheim, Mannheim, Germany, and Sigma, Deisenhofen).

(H) Localization of DegP2 in chloroplast sub-fractions and their topology in the thylakoid membranes

Intact Arabidopsis chloroplasts were isolated and purified on a Percoll gradient according to Cline (J. Biol. Chem. (1986), 261: 14804-14810). For further fractionation, the chloroplasts were broken up by osmotic shock (10 min. At 4 ° C.) in 50 mM Tris (pH 8.0) and 5 M MgCl ₂ at a chlorophyll concentration of 1 mg / ml and the thylakoid and Stroma fractions separated by centrifugation at 10,000 xg (10 min., 4 ° C). For the preparation of thylakoid lumens, isolated thylakoids were incubated in the above buffer, treated with ultrasound 10 times for 30 seconds at 4 ° C. (strength: 8.0; Misonix Inc., Farmingdale, NY, USA) and the thylakoid membranes were removed from the soluble lumen fraction separated by centrifugation at 220,000 xg (1 hour at 4 ° C). The proteins in each fraction were separated by SDS-PAGE and the position of DegP2 was determined by immunoblots.

To isolate closely fitting and not closely fitting membrane areas, the thylakoids were broken up mechanically using a "Yeda" press and the membrane fragments were separated by differential centrifugation and aqueous polymer two-phase partitioning as described by Andersson and Anderson (Biochim. Biophys. Acta 593 ( 1980), 427-440).

To isolate the peripheral thylakoid proteins, the thylakoid membranes were resuspended in 25 mM MES (pH 6.5) at a chlorophyll concentration of 0.1 mg / ml and the extrinsic membrane proteins with 0.5 M NaCl or 0.5 CaCl ₂ 30 min. extracted at 4 ° C in the dark with gentle stirring. The suspension was centrifuged at 220,000 xg (1 hour at 4 ° C) and the pellet (integral membrane proteins) and the supernatant (peripheral membrane proteins) were analyzed by SDS-PAGE and immunoblots.

The topology of the DegP2 protease was tested using a protease protection assay. Isolated thylakoid membranes were resuspended in 50 mM HEPES (pH 8.0) and 330 mM sorbitol at a chlorophyll concentration of 1 mg / ml and incubated for 30 min at 4 ° C. with 50 μg / ml trypsin. The protection of DegP2 against trypsin degradation was tested using immunoblots.

(I) Reconstitution of recombinant DegP2 with thylakoid membranes. For reconstitution studies, thylakoid membranes were isolated and 9 µg chlorophyll-containing aliquots were denatured for 10 minutes at 90 ° C (to arrest endogenous proteolytic activities). After adding enriched recombinant DegP2 (0.1 μg protein), the assays were incubated for 2 hours at 4 or 37 ° C. in 50 mM Tris (pH 9.5), 5 mM MgCl ₂ , 2 mM GTP. The breakdown of the Dl protein was investigated using SDS-PAGE and immunoblots.

To investigate light-induced degradation of the Dl protein, isolated thylakoid membranes were washed with 0.5 M NaCl to reduce endogenous DegP2 activity and then either in the dark or in strong light (5000 μmol / m ² / s) as described by Spetea et al , (PNAS USA 96 (1999), 6547-6552) for 90 minutes at 0 ° C. Aliquots of the thylakoid membranes were used for reconstitution studies and after the Dl protein had been broken down for 120 min at 37 ° and a light intensity of 10 μmol / m ² / s in the presence or absence of recombinant DegP2, immunoblots were prepared.

Example 2: Isolation of the gene encoding DegP2

The DegP / Htr family ^■ in prokaryotes (including the cyanobacteria from which chloroplasts are derived) comprises three serine-type endopeptidases (DegP (= HtrA), DegQ (= HhoA) and DegS (= HtrH or HhoB)). A homologue of the DegP of cyanobacteria in chloroplasts from Arabidopsis and pea was recently identified (DegPl) and it was shown that this protein is associated with the lumen side of the thylakoid membranes. BLAST searches of the Arabidopsis database with the conserved amino acid region of the family Deg / Htr proteases from strain PC6803 (CyanoJa7teriu-τ. Synechocystis sp.) Revealed the presence of another highly conserved homologue to the DegP / HtrA protease, the name DegP2 received. It was assumed that a gene located on Arabidopsis chromosome 2 (accession number AC005309, Protein identity AAC63648) encodes this protease.

Based on the assumed genomic sequence of DegP2, primers were designed and used in a PCR for the amplification of DegP2 cDNA from an Arabidopsis cDNA library. The DegP2 cDNA sequence contains an open reading frame (ORF) of 1821 bp, which encodes a protein with 607 amino acids (relative molecular mass: 66.8 kD). A homology comparison of the deduced amino acid sequences revealed that the overall sequence homology of DegP2 and DegPl to the Deg / Htr protease of the cyanobacteria was 44% and 48-51%, respectively. A comparison of the conserved regions of DegP2 and DegPl and with the homologous proteases of the Deg / Htr family from Synechocystis is shown in FIG. 2A. Three amino acids (S, H and D), which are involved in the catalytic activity of the serine proteases of the Deg / Htr family, are present in highly conserved positions in DegP2. DegP2 has an N-terminal transit peptide (amino acids 1-69, typical for proteins imported into plastids) and two strongly conserved domains (FIG. 2B). A catalytic domain specific for trypsin-type serine proteases has been found between amino acids 144 and 282, as well as a repeating unit of 65 amino acids (the so-called PDZ domain) between amino acids 308 and 373. The PDZ domain in eukaryotes is commonly referred to as a protein / protein -Interaction point, whereby the same function is assumed for prokaryotes. Hydropathy plots revealed that the mature DegP2 protein is a predominantly hydrophilic protein with a molecular mass of 60 kD and does not have any transmembrane domains, but a short hydrophobic segment located at the C-terminus (amino acids 440-450).

To investigate the copy number of the DegP2 gene in Arabidopsis, genomic DNA was isolated, cleaved with restriction enzymes and hybridized with DegP2 cDNA. A single band could be detected regardless of the stringency of the hybridization (FIG. 2C), which indicates that the DegP2 gene is present at a single locus in the Arabidopsis genome. Example 3: Biochemical characterization of proteolytic

DegP2 activity

To study the proteolytic activity of DegP2, it was overexpressed in a bacterial system as a fusion protein with a His tag at the C-terminus. The proteolytic activity of an E. coli extract with overexpressed DegP2 and the fractions containing recombinant DegP2 and purified by affinity chromatography were tested on a non-specific protease substrate (gelatin) (FIG. 3A). As negative controls, an E. coli extract without recombinant DegP2 (FIG. 3A) and an E. coli extract with an overexpressed non-protective protein (the class la metallothionein) were analyzed on activity gels under identical experimental conditions. The results show (FIG. 3A, lower section) that the fractions containing overexpressed DegP2 can degrade gelatin in the region of DegP2 migration. A weaker proteolytic band visible in the fractions containing DegP2 could represent an autodegradation product of DegP2. Although various proteolytic activities were visible in control fractions, the lack of gelatin degradation in the region corresponding to the DegP2 migration showed that the measured proteolysis was mediated by the recombinant DegP2 protease (FIG. 3A).

The presence of conserved catalytic amino acids in the DegP2 sequence (Figure 2A) suggested that this enzyme is a trypsin type serine endopeptidase. To confirm this assumption, various protease inhibitors were carried out in vitro (FIG. 3B). The gelatin breakdown in activity gels due to overexpressed DegP2 was completely arrested or greatly reduced in the presence of serine protease inhibitors such as 3,4-dichloroisocoumarin (DCI), aprotinin or phenylmethylsulfonate (PMSF). Inhibitors directed against cysteine, aspartate or metallo-endopeptidases did not significantly affect the proteolytic activity of DegP2.

Example 4: Studies on the chloroplast localization of DegP2 and on the thylakoid membrane topology

A polyclonal antibody against the recombinant protein was generated to investigate the subcellular localization of DegP2. The fractionation of isolated intact chloroplasts in stroma, thylakoid membrane and thylakoid lumen and then the immunoblot analysis of the fractions revealed that the overwhelming proportion of DegP2 - Protass e was associated with the thylakoid membranes, in the stroma fractions only very small amounts can be detected (Figure 4A). The presence of DegP2 in the stroma may be caused by contamination of this fraction with small membrane fragments that did not sediment during centrifugation. These results are consistent with the location predicted based on the presequence of DegP2. Further fractionation of the thylakoid membranes into tight and not tight membrane areas by means of differential centrifugation and aqueous polymer two-phase partition showed that 80-90% of this enzyme was present in the stroma lamellae and 10-20% in fractions that were enriched in non-close-fitting areas by stacks of grana consisting of grana edges and grana end membranes

(Figure 4B). The distribution of subunit A of the PSI reaction center (PSI-A) and the Dl protein was in agreement with previous data. While the overwhelming majority of the Dl protein was detected in the Grana region, the majority of PSI-A was found in the stroma lamella

(Figure 4B).

In order to investigate whether the DegP2 protease is a peripheral or integral membrane protein, the isolated thylakoid membranes were used to release extrinsic membrane proteins washed with salt. Extraction of DegP2 with 0.5 M NaCl or 0.5 M CaCl ₂ from the thylakoids showed that this protein is a peripheral membrane protein (FIG. 4C, lower section). In addition, the presence of DegP2 in the NaCl supernatant indicates that this protein is associated with the stroma side of the membranes. For reference, the fractionation pattern of the 33 kD protein from PSII's oxygen-evolving complex (PsbO) is shown (Figure 4C, lower section). PsbO is a peripheral membrane protein located on the lumen side of the thylakoid membrane. It could be shown that PsbO can be easily extracted from the membrane with 1 M CaCl ₂ , but not with 1 M NaCl.

The topology of DegP2 within the thylakoid membranes was also verified by the susceptibility to added tryps (FIG. 4D, right section). The incubation of the isolated thylakoid membranes with trypsin led to a cleavage of DegP2, which shows that this protein is not protected by the lipid bilayer and is therefore susceptible to degradation by trypsin. In contrast, PsbO was protected from tryptic degradation (Figure 4D, left section).

Example 5: The expression of DegP2 varies under different stress conditions

To examine the effect of various types of stress on the expression of the DegP2 gene, leaves of Arabidopsis were subjected to cold stress, heat shock, high salt conditions, dehydration, oxidative stress, UV-A radiation and light stress, and then the concentration of the DegP2 transcript and of the protein was examined by means of Northern (FIG. 5A) or Western blots (FIG. 5B). As a control, leaves were exposed to normal light or temperature conditions and the amount of DegP2 transcript and protein expressed under these conditions was used as a reference. after the Leaves were exposed to elevated temperatures and treatment with H ₂ 0 ₂ , only traces of the DegP2 transcript and the protein could be detected. Salt stress and dehydration also led to a reduction in the amount of DegP2 transcript (FIG. 5A). In contrast, the amount of DegP2 protein was increased two to four times under the last two conditions compared to the control sample in the thylakoid membranes. In addition, the light stress promoted the accumulation of DegP2 in the thylakoid membrane (Figure 5B).

Example 6: The physiological target of DegP2 is the photo-damaged Dl protein of the PSII reaction center

Since the degradation of the Dl protein after photoinhibition is catalyzed by a membrane-bound protease of the serine type, the investigation of the physiological goal of the DegP2 protease appeared of interest. The addition of DegP2 to isolated thylakoid membranes whose endogenous proteolytic activities had been inhibited by heating led to the specific disappearance of a 32 kD membrane protein, which could be demonstrated by SDS-PAGE and Coomassie blue staining. The 32 kD protein was not affected when an E. coli control extract was added to the reconstitution assay. In order to determine the identity of the 32 kD protein, immunoblots were carried out with a polyclonal anti-Di antibody. The band degraded during the reconstitution with purified recombinant DegP2 could be identified as the Dl protein of the PSII reaction center (FIG. 6A, left section). The typical proteolytic N-terminal 23 kD fragment of the Dl protein was also detected by immunoblots. The corresponding proteolytic C-terminal 10 kD fragment was not recognized by the anti-Dl antibody used. Immunoblots with antibodies to Lhcb2, the α subunit of Fl ATPase, and purified total PSI showed that the concentration of these proteins in the thylakoid membranes was not significant in the presence of DegP2 changed (Figure 6A, right section). This confirms the selectivity of DegP2 proteolysis.

The DegP / HtrA protease in E. coli has a chaperone function at low temperatures and a proteolytic activity at elevated temperatures. It could be shown that bacterial DegP / HtrA is proteolytically inactive below 22 ° C. To investigate whether DegP2 behaves similarly in higher plants, the reconstitution assays were carried out at 4 and 37 ° C in the presence or absence of recombinant DegP2. The results

(FIG. 6B) show that in the presence of DegP2, the Dl protein is broken down at both temperatures, but the 23 kD proteolytic fragment was only detected in assays which had been carried out at 4 ° C. The fact that this fragment is missing in assays at 37 ° C indicates that either overexpressed DegP2 was involved in a secondary cleavage or that endogenous thylakoid proteases still retained partial activity. In control assays reconstituted with E. coli extracts without recombinant DegP2 or with extracts expressing class la metallothionein, only small amounts of the 23 kD degradation fragment could be detected at 37 ° C. This supports the assumption that endogenous DegP2 denatured during the incubation period regained proteolytic activity. These results show that DegP2 actually catalyzes the primary cleavage of the Dl protein that has been denatured by heat treatment, which process is not temperature dependent. However, the low temperature led to a significant decrease in the degradation kinetics of the 23 kD proteolytic fragment

(Figure 6B), presumably by partial inhibition of the FtsH protease.

In order to investigate whether the degradation of the Dl protein by recombinant DegP2 is stimulated by the presence of GTP, the kinetics of the Dl degradation in the presence or absence of GTP were measured. The results (Figure 6C, left section) show that the degradation of the Dl protein up to two to fourfold was stimulated by adding GTP. In the absence of recombinant DegP2, the addition of GTP did not affect the DI level in heat-inactivated thylakoid membranes (FIG. 5C, right section).

Finally, an experiment was designed to investigate whether DegP2 can degrade the Dl protein in thylakoid membranes photoinhibited by strong lighting. Such treatment was believed to first induce irreversible oxidative damage, which in turn triggers a change in the conformation of the Dl protein, making sites that are normally protected from proteolysis now accessible. By using thylakoid membranes pretreated with darkness or strong light, in which endogenous proteolytic activity directed against the Dl protein had been partially removed by NaCl washing steps, it could be shown that added recombinant DegP2 can recognize and degrade light-damaged Dl protein (FIG. 6D , upper and middle section). In the absence of recombinant DegP2, only traces of the proteolytic 23 kD fragments could be detected and these were probably related to the activity of the remaining endogenous DegP2. In contrast, the Dl protein in the dark control sample was not susceptible to proteolytic degradation by overexpressed DegP2. A turnover of the reaction center counterpart of the Dl protein, namely the D2 protein, and a slight degradation of CP43 under strong photoinhibitory conditions have already been reported. It was therefore investigated whether the proteolytic activity of DegP2 is also directed against these two proteins. Immunoblot analyzes showed that neither the D2 protein nor C43 were degraded by DegP2 protease under the conditions tested.

The following clone was deposited with the German Collection for Microorganisms and Cell Cultures GmbH, Mascheroder Weg, Braunschweig (DSMZ) in accordance with the Budapest Treaty on January 26, 2001: Escherichia coli DegP2: DSM 14011

Claims

claims

1. DNA sequence encoding the protease DegP2 with the amino acid sequence shown in FIG. 1B.

2. DNA sequence according to claim 1, which comprises the coding region of the DNA sequence shown in Figure 1.

3. DNA sequence encoding a protein with the biological properties of the protease DegP2,

(a) which differs from the DNA sequence of claim 2 in the codon sequence due to the degeneration of the genetic code;

(b) which hybridizes to the DNA sequence of claim 2 or 3 (a) under stringent conditions;

(c) which has at least 65% homology to the DNA sequence of claim 2 or 3 (a); or

(d) which is a fragment, an allelic variant or another variant of the DNA sequence of claims 2 or 3 (a) to 3 (c).

4. Ribozyme, characterized in that it is complementary to the DNA sequence according to any one of claims 1 to 3 and can specifically bind to the mRNA transcribed by this DNA sequence and can cleave it, whereby the synthesis of the encoded by this DNA sequence Protein is reduced or inhibited.

5. Antisense RNA, characterized in that it is complementary to the DNA sequence according to one of claims 1 to 3 and can bind specifically to the mRNA transcribed by this DNA sequence, thereby the synthesis of the protein encoded by this DNA sequence is reduced or inhibited.

6. vector, the DNA sequence according to one of claims 1 to 3 or the ribozyme according to claim 4 or the antisense RNA according to Claim 5 containing coding DNA.

7. host cell transformed with the expression vector of claim 6.

8. DegP2 protease or protein with their biological activity which (which) (a) is encoded by the DNA sequence according to one of claims 1 to 3 or (b) shows the same functionality in an in vitro assay.

9. A method for producing a DegP2 protease or a protein with its biological activity, which comprises culturing the host cell according to claim 7 under suitable conditions and recovering the protein from the cell or the culture medium.

10. Antibody that specifically binds to the DegP2 protease or a protein with its biological activity according to claim 8.

11. Use of the DNA sequence according to one of claims 1 to 3, the expression vector according to claim 6 or the DegP2 protease or the protein with its biological activity according to claim 9 for protecting plants from damage induced by light, for faster recovery of plants of light damage that has already occurred, to protect against high salt concentrations, dehydration or oxidative stress.

12. Use of the ribozyme according to claim 4, the antisense RNA according to claim 5, the expression vector according to claim 6 or the antibody according to claim 10 for reducing or suppressing the degradation of the photo-damaged Dl protein.

13. Kit containing the DNA sequence according to one of claims 1 to 3 and / or the antibody according to claim 10.

14. Transgenic plant containing the vector according to claim 6.

15. Transgenic plant according to claim 14, which comes from the group Gramineae, Chenopodia, legume, Brasicaceae, Solanaceae, fungi, moss or algae.

16. The transgenic plant according to claim 15, which is wheat, barley, rice, corn, sugar beet, sugar cane, potato, rapeseed, mustard, beet, flax, pea, bean, lupine or tobacco.