DE19853778C1 - DNA sequences encoding a glutamate / malate translocator, plasmid bacteria, yeast and plants containing this transporter - Google Patents

Abstract

The present invention relates to DNA sequences from sorghum bicolor (millet), Flaveria bidentis and Spinacia oleracea (spinach), which contain the coding region of a glutamate / malate translocator, the introduction of which into a plant genome involves the formation and transmission of carbon frameworks for the fixation of Nitrogen and the forwarding of the assimilated nitrogen in transgenic plants changed, plasmids, yeasts and bacteria, containing these DNA sequences, as well as transgenic plants in which, by introducing the DNA sequences, changes in the activity of the glutamate / malate translocator and thus changes in nitrogen - and carbon metabolism are caused. Furthermore, the invention relates to transgenic plants which are affected by the change in the activity of the glutamate / malate translocator in their ability to photorespiration. The invention also relates to the use of the described sequences of the glutamate / malate translocator for identifying related translocators from plants (angiosperms, gymnosperms and algae) by hybridization with low stringency or by PCR techniques, and to the use of the glutamate / malate translocators as a target ("Target") for herbicides.

Description

The present invention relates to DNA sequences from sorghum bicolor (millet), Flaveria bidentis and Spinacia oleracea (spinach), which are the coding region of a Contain glutamate / malate translocators, their introduction into a herbal Genome the formation and transfer of carbon skeletons for the Fixation of nitrogen and transfer of the assimilated nitrogen in transgenic plants altered, containing plasmids, yeasts and bacteria DNA sequences, as well as transgenic plants in which the introduction of DNA Sequences changes in the activity of the glutamate / malate translocator and thus causing changes in nitrogen and carbon metabolism become. The invention further relates to transgenic plants which are characterized by Change in the activity of the glutamate / malate translocator in its ability to Are affected by photorespiration. The invention also relates to the use of the described sequences of the glutamate / malate translocator for identification related translocators from plants (angiosperms, gymnosperms and Algae) by hybridization with low stringency or by PCR techniques, and the use of the glutamate / malate translocators as a target for Herbicides.

Only plants, bacteria and yeast are used to transfer inorganic Nitrogen (nitrate nitrogen) in organically bound nitrogen on a large scale (in usually in the form of amino acids) by reductive amination of organic Capable of carbon compounds. The rest of the busy world, in particular Farm animals and humans is more organic on plants as primary suppliers Nitrogen compounds. Assimilation of inorganic nitrogen in plants by binding to organic carbon depends on availability on nitrogen, carbon frameworks and energy.

The nitrogen supply to the plant must be influenced by fertilization. The energy for nitrogen assimilation comes from the light reaction of photosynthesis or in roots and other non-green tissues from the dissimilation and is under no limiting factor under normal field conditions.

2-Oxoglutarate (α-ketoglutarate) is the primary one according to current knowledge Reduced nitrogen acceptor in glutamine synthetase / glutamine-2 Oxoglutarate aminotransferase (GOGAT) reaction. In this sequence of reactions nitrogen (ammonium nitrogen) initially reduced by glutamine synthetase transferred to the amino acid glutamate while consuming energy. It arises  Glutamine. In a subsequent reaction, the glutamate oxoglutarate catalyzes Aminotransferase (GOGAT; glutamate synthase) using Reduction equivalents the transfer of an amino group from glutamine to 2- Oxoglutarate (transamination). Two molecules of glutamate are formed. In the The sum of one molecule of glutamate, one molecule of 2-oxoglutarate, one Molecule ammonium and ATP as energy equivalent and reduced ferredoxin as a reduction equivalent two molecules of glutamate, ADP and oxidized ferredoxin educated. It can be seen that a net gain from one molecule of organic Nitrogen compound (glutamate) each time the reaction cycle is run through the reductive amination of a molecule 2-oxoglutarate is achieved.

The entire reaction sequence of the glutamine synthetase / GOGAT reaction is in Stromal of the plastids of plants localized. These organelles are of two Enclosed lipid bilayer membranes, their outer molecular sieve character and allows connections up to a size of approx. 10 kDa to pass freely (Flügge and Benz, 1984, FEBS Lett. 169: 85-89). The inner membrane is permeable to some smaller compounds such as water, carbon dioxide, oxygen and nitrite, but not for larger charged molecules such as glutamate or malate.

This key compound of the glutamine synthetase / GOGAT reaction must come from the Cytosol of the plant cell through a specific translocator through the inner Plastid envelope membrane are moved into the plastid stroma. The transport of 2-oxoglutarate in the plastids is exchanged with malate from the Plastids through the 2-oxoglutarate / malate translocator. That in this process the cytosol exported malate is by a second translocator, the Glutamate / malate translocator, transported back in exchange for glutamate. It it should be noted that the 2-oxoglutarate / malate translocator, in contrast to the glutamate / malate translocator, not the transport of glutamate (or also Aspartate) can catalyze. As a result, without a net transport of Malate, which circulates over both translocator systems ("double translocator", Woo et al., 1987, Plant Physiol. 84: 624-632; Flügge et al., 1988, Planta 174: 534-541), 2- Oxoglutarate imported into the chloroplast and the end product of the Glutamine synthetase / GOGAT reaction, glutamate, exported. Glutamate is used in the Plant as a preferred amino group donor in a number of Transamination reactions, for example in the biosynthesis of amino acids Alanine or Phenylalanine etc. Most nitrogenous compounds in the Plant like amino acids, nucleic acids or alkaloids need in their Biosynthetic pathway initially glutamate as the primary amino group donor. Farther is an important form of transport for organically bound nitrogen within the plant and as such is incorporated into the plant's lead tissue (the  Phloem). The most common amino acids in phloem juice are usually Glutamate, glutamine and aspartate.

Transporters are in the allocation of the photoassimilate (i.e. primarily sugar and Amino acids) from the "source" to the "sink" organs at a crucial point integrated - like the chloroplastic triose phosphate / phosphate translocator, the Sucrose translocator (over which the sieve tubes with the transport form of the Photoassimilates loaded sucrose) and the glucose-6- phosphate / phosphate translocator of amyloplasts. Activity changes a specific membrane transporter can have a big impact on the Have metabolic performance of plants. So it could be shown recently that the effectiveness of photosynthetic carbon reduction (light reaction and Calvin cycle) very much due to the activity of the chloroplastic Triose phosphate / phosphate translocator can be influenced, the Transport of the fixed carbon catalyzed by the chloroplasts. The Inhibition of this transporter in planta via the expression of a corresponding one "antisense" mRNA leads to a reduced export of the primary Photoassimilates (triose phosphate, 3-phosphoglycerate), which in these circumstances Within the chloroplast is derived in the biosynthesis of starch massive build-up (Riesmeier et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6160-6164; Heineke et al., 1994, Planta 193: 174-180). This translocator protein represents consequently represents a bottleneck in the carbon metabolism of the "source" tissue. The reduction of sucrose transport activity via an "antisense" Inhibition of the sucrose responsible for transport into the sieve tubes Transporters (Riesmeier et al., 1992, EMBO J. 11: 4705-4713) leads to one drastic phenotype of plants: they are much smaller, the leaves are damaged and the plants hardly form tubers in the case of potatoes d. H. the supply of the "sink" tissue with photoassimilates is greatly reduced (Riesmeier et al., 1994, EMBO J. 13: 1-7).

Likewise, the reduction in the transport capacity for 2-oxoglutarate via the plastid envelope membrane by antisense repression of the 2-oxoglutarate / malate translocator mRNA in tobacco plants leads to a drastic phenotype. The plants have a compressed, very slow growth with late flowering. The leaves showed fading along the guide bundle, the intercostal fields were still colored green. The plants accumulated ammonium and chloride, but showed drastically reduced nitrate levels (own, unpublished observations). Since the glutamate / malate translocator is integrated in the same biochemical pathway in a central position, a similar effect of repression of this transporter on the phenotype of plants can be assumed. Further evidence for this assumption is an observation by Somerville (1983, 1985), in which an EMS mutant of the glutamate / malate translocator in Arabidopsis is described. This is not viable under ambient CO ₂ conditions because it cannot carry out a complete photorespiratory route (Somerville and Ogren, 1983, Proc. Natl. Acad. Sci. USA 80: 1290-1294; Somerville and Somerville, 1985, Plant Science Letters 37 : 217-220). However, the mutant described has so far not been used to identify and clone a glutamate / malate translocator.

As described above, the glutamate / malate translocator takes the Plastid envelope membrane play a key role in the intracellular distribution of the the nitrogen fixation involved amino group acceptors and donors. It would be possible to clone this translocator and with the help of the sequence obtained Modulating the activity of the translocator in plants opened the way to plants with a changed carbon / nitrogen ratio. This means, that plants could be produced which, for. B. less carbohydrates (sugar, Starch, fats) and more organic nitrogen compounds (protein, amino acids, Alkaloids) contained. On plants with such modified ingredients urgent need because the vast majority of people on earth are up plant-based diet, with the consequence of a constant Protein deficiency in these strata of the population. Likewise, in the Animal feeding in developed countries is increasing animal and fish meal Cattle feed added to improve the protein supply for breeding animals. Forage crops with a higher protein content would certainly be the better alternative here, especially with a view to those caused by feeding animal meal Problems such as BSE etc. An influence of overexpression of the 2-oxoglutarate / malate Translocators in tobacco on the C / N ratio of tobacco plants could already be shown (Weber, 1995, dissertation, University of Würzburg). Since the Glutamate / malate translocator has a wider substrate spectrum than the 2- Oxoglutarate / malate translocator, this is influencing the C / N ratio in transgenic plants with altered expression of this transporter presumably even more pronounced.

The drastic phenotype of a mutation in the glutamate / malate translocator (see above) makes this protein interesting as a target for herbicides. An inhibitor this transporter should be usable as a total herbicide. As for one further enzyme of the photorespiratory pathway could be shown (Glutamine synthetase, GS) inhibits this enzyme by a herbicide (Phosphinotricine, BASTA) to kill the treated plants. By Introduction of an enzyme activity that metabolizes the possible inhibitor and This could make targeted resistance to this drug ineffective be generated. Likewise, this would be through a targeted change in the DNA or  the resulting amino acid sequence of the transporter possible. A such a change would change the binding properties of the fuel and make it ineffective without the transport function affect. To find such an inhibitor and to find it Possible sequence changes of the transporter protein can be done by us described system of reconstitution of recombinant proteins in proteoliposomes with subsequent function measurement (see embodiment 2).

We have recently succeeded in developing one for the plastid 2-oxoglutarate / malate Cloning translocator-encoding cDNA (Weber et al., 1995, Biochemistry 34: 2621-2627). The primary sequence of this translocator differs fundamentally from the sequences of the known 2-oxoglutarate / malate carriers from the Mitochondria from bovine hearts and from human mitochondria (Runswick et al., 1990, Biochemistry 29: 11033-11040; Iacobazzi et al., 1992, DNA Seq. 3 (2): 79-88). These transporters play an important role in mitochondrial dicarboxylic acid Metabolism (including malate / aspartate shuttle, oxoglutarate / isocitrate shuttle) and are belongs to the family of mitochondrial metabolite transporters that are close to each other are related. For most mitochondrial carriers, as well as for the 2- Oxoglutarate carriers could be shown to be without them Organelle directing addressing sequence ("targeting" sequence) in the Mitochondrial membrane can be incorporated (Runswick et al., 1990, Biochemistry 29: 11033-11040). It can therefore be assumed that the "targeting" information is mature Carrier protein is included.

In contrast, plastid translocators need for a correct one Addressing an appropriate pre-sequence to the inner plastid membrane ("Targeting" sequence), which attaches the mature protein correctly to the plastids conducts (review article: Keegstra et al., 1989, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 471-501; Lubben et al., 1988, Photosynth. Res. 17: 173-194; Flugge, 1990, J. Cell Sci. 96: 351-354). In addition to that necessary for "plastid addressing" There is a presequence in the mature part (the part of the protein that is released after the Pre-sequence by a specific protease remains) of the plastid envelope membrane Proteins still further information necessary for the specific insertion of the proteins in the membrane are responsible and a transport of the envelope membrane proteins over the envelope membrane into the plastid stroma or, in the case of chloroplasts, the Prevent thylakoid membrane (Knight and Gray, 1995, Plant Cell 7: 1421-1432 and own investigations: Brink et al., 1995, J. Biol. Chem. 270: 20808-20815). Own Work also showed the example of a mitochondrial carrier, the ADP / ATP Transporters that this protein is not or only with a very low efficiency Plastids can be directed and built into the envelope membrane. Yourself  Hybrid protein consisting of a plastid presequence (the information for the Containing plastid addressing) and this mitochondrial carrier compared to the authentic protein a hardly increased incorporation in the Plastid sheath membrane (Silva-Filho et al., 1997, J. Biol. Chem. 272: 15264-15269; unpublished observations). Since the protein / lipid Interaction is important and the plastid envelope membrane in its Lipid composition fundamentally different from that of other cell organelles Differentiates bacteria (Joyard et al., 1991, Eur. J. Biochem. 199: 489-509), it is very unlikely to be a, albeit minor, insertion of a heterologous Proteins, then is also functional, d. that is, a transporter from other systems at all in a conformation and orientation that corresponds to its function can be built into the plastid envelope membrane.

Thus, according to the current state of the art, it is not possible with help known plastid "targeting" sequences e.g. B. mitochondrial or prokaryotic dicarboxylic acid transporters functionally inside Integrate plastid envelope membrane. At the current state of the art, it is more promising for the construction of the plants described above necessary DNA sequence by cloning the authentic plastid Obtain glutamate / malate translocators.

Although this transport system also catalyzes the transport of malate, it is Primary structure is believed to be very different from that of the recent one identified 2-oxoglutarate / malate translocator. Try using the translocator a low-stringent screening of a plant cDNA library a probe derived from the 2-oxoglutarate / malate translocator identifying remained unsuccessful, as was identifying the translocator on a biochemical characterization, purification and isolation of the Glutamate / malate translocators (own observations).

A molecular biological approach was therefore chosen to identify the translocator, which is based on the differential expression of a particular mRNA species in different cDNA populations (differential screening; embodiment 1). In contrast to C ₃ plants (e.g. potato, tomato), C ₄ plants, such as B. maize, millet (Sorghum bicolor) or Flaveria bidentis two different leaf cell types, mesophyll and bundle sheath cells, which are involved in the production of the photoassimilate. In the mesophyll cells, the primary fixation of atmospheric carbon dioxide takes place with the formation of a C4 body (e.g. malate), which is then transported to the bundle sheath cells. After the carbon dioxide has been released from the C4 body, the actual biosynthesis of the photoassimilate takes place there. CDNA libraries from mesophyll cells and bundle sheath cells from Sorghum bicolor were produced and it was checked which mRNAs are specifically expressed only in the bundle sheath cells. In this way it was possible to identify a partial cDNA clone which had little homology with the 2-oxoglutarate / malate translocator (Wyrich et al., 1998, Plant Mol. Biol. 37: 319-335). The corresponding cDNA insert was used as a probe for the subsequent screening of a cDNA library from Sorghum bicolor and a cDNA clone could be isolated which contained the complete cDNA (2088 base pairs) for the corresponding precursor protein with a molecular mass of 59 kDa .

A comparison of the protein sequence derived from the cDNA with sequences of the Databases showed little similarity to the well-known 2- Oxoglutarate / malate translocator (approx. 50%). It could therefore be assumed that this cDNA encodes a transporter whose activity is different from that of the 2-oxoglutarate / malate translocator. Homology was also evident a genomic DNA sequence from Arabidopsis thaliana, which is available internationally pursued sequencing projects. This sequence is on Chromosome 5 of Arabidopsis thaliana localized and also shows a low one Homology to the sequence of the 2-oxoglutarate / malate translocator from spinach of approx. 50%. As part of the genome sequencing project, only the Homology referred, but the function was not determined.

The function of the transport protein encoded by the cDNA was determined in a heterologous expression system, the split yeast Schizosaccharomyces pombe. Since, based on our own observations, transport proteins cannot be produced heterologously from monocotyledonous plants, cDNA sequences from the C ₄ plant Flaveria bidentis and from the C ₃ plant spinach which corresponded to the sequence of the Sorghum bicolor cDNA were first isolated. The cDNA insert from Flaveria bidentis was then introduced into a eukaryotic yeast expression vector and cells of the fission yeast Schizosaccharomyces pombe were transformed with this construct. Transformants expressing the corresponding plasmid were isolated. Transport experiments carried out with membrane fractions of the yeast transformants reconstituted in artificial membranes showed that this translocator catalyzes the transport of malate in exchange for glutamate (or aspartate). In contrast to the 2-oxoglutarate / malate translocator, this translocator also accepts amino acids (glutamate and aspartate) as counter-exchange substrates (working examples 2 and 3). The transporter was given the name glutamate / malate translocator.

The present invention thus provides DNA sequences which consist of a plant genome and for a plastid glutamate / malate Code translocator, the information contained in the nucleotide sequence at Introduction and expression in plant cells to form a ribonuclein  acid leads and a glutamate / malate translocator via this ribonucleic acid Activity can be introduced into the cells or an endogenous glutamate / malate Translocator activity can be suppressed.

The invention relates in particular to the DNA sequences from sorghum bicolor (millet) with the nucleotide sequence Seq-ID No .: 1, a DNA sequence from Flaveria bidentis with the nucleotide sequence Seq-ID No .: 2 and a DNA sequence from Spinacia oleracea (spinach) with the nucleotide sequence Seq-ID No .: 3 ( Fig. 1).

Another object of the invention are DNA sequences with the under Seq. ID No .: 1-3 called DNA sequences or parts thereof or derivatives which are derived from these sequences by insertion, deletion or substitution, hybridize and code for a plastid protein that has biological activity a dicarboxylic acid translocator, in particular a glutamate / malate translocator owns.

In addition, the present invention relates to the use of the DNA sequences according to the invention or parts thereof or derivatives which by Insertion, deletion or substitution are derived from these sequences for Transformation of pro- and eukaryotic cells. In order to express the The glutamate / malate translocator in transformed cells can ensure that DNA sequence according to the invention are introduced into plasmids and thereby Controls for expression in prokaryotic or eukaryotic Cells can be combined (see working examples 2 and 4). Such Control elements are, on the one hand, transcription promoters and, on the other hand Transcription terminators. With the plasmids, eukaryotic cells can are transformed with the aim of expressing a translatable Messenger ribonucleic acid (RNA), which is the synthesis of a plastid glutamate / malate Translocators allowed in the transformed cells, or with the aim of Expression of a non-translatable, inversely oriented ("antisense"), Messenger ribonucleic acid, which is the synthesis of endogenous glutamate / malate Prevents translocators. For this purpose, shorter fragments of the DNA sequence according to the invention or DNA sequences with a relatively high Degree of homology (more than about 65% homology) can be used. Likewise can the expression of endogenous dicarboxylic acid translocators by the expression a ribozyme constructed for this purpose using the DNA sequences according to the invention are inhibited. By expressing a RNA according to the sequence of a plant according to the invention Glutamate / malate translocator is a change in the plant Nitrogen metabolism possible, the economic importance of which is that an improvement in the transfer of glutamate from the cytosol of the plastids to a change in the ratio of carbohydrates (sugar, starch, org.  Acids) and fats in favor of nitrogen compounds (amino acids, proteins, possibly alkaloids) takes place. Plants can thus be created that are richer in are valuable protein, but lower in carbohydrates and Have fats. This modification increases the nutritional value of plants and thus their economic value. Furthermore, the heterologous expression of the described sequence in gap yeasts (Examples 2 and 3; further: Loddenkötter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2155-2159) Structure-Functional Studies of Glutamate / Malate Translocators, which ultimately lead to the development of a specific inhibitor for this protein could lead to herbicide development think because the inhibition of a key protein in the Metabolism would inevitably be lethal for the plant. Furthermore, these Structure-function studies basis for a directed mutagenesis of the Substrate binding site of the translocator to the substrate specificity of the Targeted changes to transporters.

There are already processes for the genetic modification of dicotyledons and monocotyledons Plants known (Gasser and Fraley, 1989, Science 244: 1293-1299; Potrykus, 1991, Ann. Rev. Plant Mol. Biol. Plant Physiol. 42: 205-225). For the expression of coding sequences in plants must be transcriptional regulatory elements. Such elements, promoters are known (et al. Koster-Töpfer et al., 1989, Mol. Gen. Genet. 219: 390-396). Furthermore, the coding regions must have a transcription termination signal provided so that they can be transcribed correctly. Such elements are also described (Gielen et al., 1989, EMBO J. 8, 23-29). The Transcriptional start area can be both native or homologous as well as alien or be heterologous to the host plant. Termination areas are against each other interchangeable. The DNA sequence of the transcription start and - Termination regions can be produced synthetically or obtained naturally or a mixture of synthetic and natural DNA components contain. To prepare for the introduction of foreign genes into higher plants a large number of cloning vectors are available which are a replication signal for E. coli and a marker containing a selection of the transformed cells allowed. Examples of vectors are pBR322, pUC series, M13 mp series, pACYC 184 etc. Depending on the method of introducing desired genes into the plant additional DNA sequences may be required. Are z. B. for the Transformation of the plant using the Ti or Ri plasmid must at least the right boundary, but often the right and left boundaries of the Ti and Ri plasmid T-DNA can be added as a flank region to the genes to be introduced. The use of T-DNA for the transformation of plant cells is intensive  examined and adequately described (Hoekema, in: The Binary Plant Vector System, offset printing Kanters B-V. Ablasserdam, 1985, Chapter V; Fraley et al., Critic. Rev. Plant Sci. 4: 1-46; An et al., 1985, EMBO J. 4: 277-287). Is the one introduced Once DNA is integrated in the genome, it is usually stable there and remains in the progeny of the originally transformed cell. It contains usually a selection marker that unites the transformed plant cells Resistance to biocides or antibiotics such as kanamycin, bleomycin or Hygromycin confers. The individually introduced marker therefore becomes the selection allow transformed cells versus cells that lack the inserted DNA. For the introduction of DNA into a plant host cell stand next to the Transformation using agrobacteria has many other techniques available. These techniques include protoplast transformation, microinjection of DNA, electroporation as well as ballistic methods. From the transformed plant material can then be in a suitable Whole plants can be regenerated. The plants thus obtained can then be detected using conventional molecular biological methods the imported DNA can be tested. These plants can be grown normally and with plants that have the same transformed genetic makeup or others Possess inheritance, to be crossed. The resulting hybrids Individuals have the corresponding phenotypic properties. The DNA sequences according to the invention (or parts of this sequence or derivatives this sequence) can also be introduced into plasmids and thereby Control elements for the expression in cells of fission yeasts can be provided (see Embodiment 2). The introduction of the glutamate / malate translocator leads to a significant increase in through reconstitution into artificial liposomes measurable activity of the glutamate / malate translocator in the recombinant Yeast cells. Recombinant yeast cells have a much higher malate Transport activity demonstrable. It is therefore conceivable with the help of the described DNA sequence (or parts or derivatives of this sequence) to yeast cells change that they have a changed protein content. Here would come Advantage of a plastid translocator to carry from plants, not that endogenous regulatory and compartment addressing mechanisms of Subject to split yeasts. These strains would be especially for the Feed industry of paramount importance.

The DNA sequences according to the invention (or derivatives or parts of this sequence) can also be introduced into plasmids which have a mutagenesis or a Sequence change by recombination of DNA sequences in allow prokaryotic or eukaryotic systems. This allows the  Specificity of the glutamate / malate translocator can be changed e.g. B. towards an affinity for other dicarboxylic acids.

Likewise, the glutamate / malate translocator could be insensitive to specific herbicides can be achieved. With the help of standard procedures (Sambrock et al., 1989, Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, USA) can base changes and / or base deletions made and / or synthetic or natural sequences added become. Adapters can be used to connect the DNA fragments to one another or left can be added. Manipulations can also be the appropriate Provide restriction sites or remove the unnecessary DNA be used. Wherever insertions, deletions or substitutions such. B. Transitions or transversions can be considered, in vitro mutagenesis, "primerrepair", restriction or ligation can be used. As a method of analysis are generally sequence analysis, restriction analysis and others biochemical-molecular biological methods such. B. the expression of modified protein in fission yeast and the measurement of the modified Transport properties in artificial liposomes (see working examples 2 and 3; further: Loddenkötter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2155-2159; such as Fischer et al., 1997, Plant Cell, 9: 453-462; Kammerer et al. 1998, Plant Cell 10: 105-117) or the measurement of the modified transport properties on in transgenic Plants expressed protein using a recent one from us for this purpose developed method (Flügge and Weber, 1994, Planta, 194: 181-185) applied. The DNA sequence according to the invention (or parts or derivatives of this sequence) can be used according to standard procedures (especially hybridization Screening of low stringency cDNA libraries below Use of the DNA sequence according to the invention or parts of the DNA sequence as a probe or the creation of probes for stringent and low-stringent Screening strategies by deriving degenerate and / or not degenerate primers from the DNA sequence according to the invention for PCR Experiments with DNA or cDNA from other plants from the genome of Isolate plants-like sequences that code for proteins. The same Transport dicarboxylic acids.

The DNA sequence according to the invention contains in the translated protein regions which are in are able to specifically target the protein synthesized on ribosomes in the cytoplasm To direct plastids and the appearance of the protein in others To prevent membrane systems of the cell. This is in contrast to the known mitochondrial 2-oxoglutarate translocators, which such Addressing sequence not included. The range of proteins that this through DNA sequence encoded protein according to the invention directed to plastids, lies  within the first hundred amino acids of the protein, is for that Transport function of the protein is not necessary and will be more successful after Insertion of the protein into the plastid envelope membrane removed. By exchanging this "Plastid targeting" sequence against one of the known "targeting" sequences e.g. B. for Mitochondria could translocate the protein into another membrane system direct eukaryotic cells and could possibly be there Change transport properties across the respective membrane. Likewise, could the "plastid targeting" sequence of the glutamate / malate translocator or endogenous Areas of the mature protein are used to extract foreign proteins (e.g. bacterial Transport proteins or transporters from yeast) into the plastids or into the To direct plastid envelope membrane of plant cells.  

For a better understanding of the embodiment on which this invention is based the most important processes used are explained below.

1. Cloning procedure

For cloning the cDNAs coding for the glutamate / malate translocator Sorghum bicolor and Flaveria bidentis became the Phage Lambda ZAPII and the Phagemid pBluescript II KS (pBSC) (Short et al., 1988, Nucl. Acids Res. 16: 7583-7600) used.

The vector SAP-E (Truenit et al., 1996, Plant Cell 8: 2169-2182) was used.

For the plant transformation, the gene constructions in the binary Vector pBinAR (Höfgen and Willmitzer, 1990, Plant Sci. 66: 221-230) cloned.

2. Bacterial and yeast strains

For the pBluescriptKS (pBSC) phagemid as well as for SAP-E and pBinAR constructs the E. coli strain DH5α (Hanahan et al., 1983, J. Mol. Biol. 166: 557-580) used.

The transformation of the pBinAR constructs in tobacco plants was carried out using the Agrobacterium tumefaciens strain C58C1, pGV2260 (Bevan, 1984, Nucl. Acids Res. 12: 8711-8720).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred to the agrobacteria by direct transformation by the method of Höfgen and Willmitzer (1988, Nucl. Acids Res. 16: 9877). The Plasmid DNA transformed Agrobacteria was by the method of Birn boim and Doly (1979, Nucl. Acids Res. 7: 1513-1523) isolated and after suitable Restriction cleavage by gel electrophoresis for correctness and orientation analyzed.  

4. Plant transformation

15 small ones were wounded with emery paper and scalpel per transformation Leaves of a tobacco sterile culture placed in 10 ml MS medium with 2% sucrose, which 100 µl of a transformed agro grown under strict selection bacterium tumefaciens overnight culture. After 15 minutes of light The leaves were shaken on MS medium with 1.6% glucose, 2 mg / l Zeatinribose, 0.02 mg / l naphthylacetic acid, 0.02 mg giberellic acid, 500 mg / l Betabactyl®, 100 mg / l kanamycin and 0.8% Bacto agar. To one week incubation at 25 ° C and 3,000 lux illuminance Beta-lactyl concentration in the medium reduced by half.

Filings

On November 12, 1998 the following phagemids in the form of E. coli strains containing these phagemids were deposited with the German Collection of Microorganisms (DSM) in Braunschweig, Federal Republic of Germany:
Phagemid pSbGMT1 (GMT1 sequence from Sorghum bicolor) (DSM 12480)
Phagemid pFbGMT1 (GMT1 sequence from Flaveria bidentis) (DSM 12478)
Phagemid pSoGMT1 (GMT1 sequence from Spinacia oleracea) (DSM 12479)

Embodiment 1 Isolation and cloning of those coding for the glutamate / malate translocator cDNAs

Poly (A ⁺ ) RNA was isolated from leaves of Sorghum bicolor, converted into double-stranded cDNA using a standard cDNA synthesis kit and cloned into the vector Lambda-ZAPII (Wyrich et al., 1998, Plant Mol. Biol. 37 : 319-335). In parallel, mesophyll and bundle sheath cells were prepared by partial digestion with pectinases and cellulases from leaves of Sorghum bicolor, and poly (A ⁺ ) RNA from them. A hybridization probe was produced from the mesophyll or bundle sheath RNA using reverse transcriptase and radioactive nucleotides, with which the cDNA library was screened differentially. The screening led, among other things, to the isolation of an approximately 900 bp cDNA (HHU51) which showed similarities to a spinach cDNA which codes for the 2-oxoglutarate / malate translocator (Weber et al., 1995, Biochemistry 34: 2621 -2627) and which is expressed specifically in bundle sheaths in Sorghum bicolor (Wyrich et al., 1998, Plant Mol. Biol. 37: 319-335). In order to isolate full-length clones for the translocator partial sequences encoded in HHU51, the cDNA library was screened with the cDNA clone HHU51 as a hybridization probe. Lambda clones that reacted positively were cleaned using standard methods. The pBluescript phagemid with the cDNA insert coding for the glutamate / malate translocator was released by in vivo excision (Short & Sorge, 1992, Meth. Enzymol. 216: 495-208). The clones were analyzed by determining the DNA sequence (dideoxy method: Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) and from this DNA sequence the primary structure of the glutamate / malate translocator ( Clone pSbGMT1) determined. The sorghum bicolor (millet) cDNA was then used to isolate the corresponding cDNA clones from Flaveria bidentis (clone pFbMGT1) and Spinacia oleracea (spinach).

Embodiment 2 Expression of the glutamate / malate translocator from Flaveria bidentis in the split yeast Schizosaccharomyces pombe

The phagemid pBluescript (pBSC) containing the insert coding for the Glutamate / malate translocator, was located on both ends located EcoRI interfaces linearized with the endonuclease EcoRI and so fragment obtained into the unique EcoRI cloning site of the yeast Expression vector SAP-E (Truenit et al., 1996, Plant Cell 8: 2169-2182) cloned. To Amplification of the construct (SAP-GMT1) in E. coli was performed in the plasmid LiCl / PEG made competent (Ito et al., 1983, J. Bact. 153: 163-168) leucine synthesis deficient S. pombe cells are transformed. Transformants were selected selected on minimal medium without leucine, since the SAP GMT1 construct the Gives yeast cells the ability to grow on leucine-free media.

Embodiment 3 Measurement of glutamate / malate translocator activity in recombinant yeast lines

Yeast cells transformed with the SAP-GMT1 plasmid were placed in minimal medium attracted to an optical density at 600 nm of 1.0 and by centrifuge gation at 3,000 × g for 5 min. The cells were then removed by vigor shake with 1/2 vol (based on the cells) broken glass beads and Glass beads and cell fragments are separated by centrifugation (600 g for 1 min).  

The supernatant was adjusted to a concentration of 0.5% (weight / volume) Triton X-100, the same volume of liposomes was added and the resulting proteoliposomes were immediately frozen in liquid nitrogen. The liposomes were previously prepared by sonicating soybean phospholipid (20 mg / ml) for 10 min at 4 ° C. in the presence of 200 mM tricine-NaOH (pH 7.6), 40 mM malate and 60 mM potassium gluconate. After thawing the proteoliposomes and sonicating the suspension again with 10 pulses of 1 s each, the proteoliposomes were removed from the surrounding medium by size exclusion chromatography on Sephadex G-25, previously with 10 mM tricine-NaOH (pH 7.6), 100 mM sodium gluconate and 50 mM potassium gluconate had been equilibrated, separated. The eluted proteoliposomes were used to measure malate transport activity. The measurement was carried out according to the "inhibitor-stop" method described by Menzlaff and Flügge (1993, Biochim. Biophys. Acta 1147: 13-18). The malate transport activity in the SAP-GMT1 transformants was compared with the malate transport activity of transformants that were only transformed with the vector SAP without the GMT1 insertion. It was shown that the malate transport activity in the SAP-GMT1 transformants (measured in pmol transported ¹⁴ C-malate / mg protein per minute) was many times higher than in the SAP control transformants. It could also be shown that, in contrast to the already known 2-oxoglutarate / malate translocator, the glutamate / malate translocator is able to catalyze the transport of amino acids (glutamate / aspartate) in exchange for malate (see Table 1).

Embodiment 4 Transformation of plants with a construction for overexpression of the Coding region of the glutamate / malate translocator

From the phagemid pBluescript-GMT1 (pBSC-SoGMT1), which inserted the cDNA for the glutamate / malate translocator from spinach (see exemplary embodiment 1), the insert was isolated by restriction digestion with EcoRV and SmaI and in the vector pBinAR (Höfgen and Willmitzer, 1990, Plant Sci. 66: 221-230) of the previously the enzyme SmaI had been cloned. After amplification of the resulting construct pBinAR-GMT1 in E. coli were the construct in Agrobacteria are transformed and then used to infect leaf segments of Tobacco and potato used.

The transformants obtained were analyzed using Southern blot analyzes investigated the presence of the intact, non-rearranged chimeric gene. With help the "whole sheet reconstitution method" (Flügge and Weber, 1994, Planta, 194: 181-185) the malate transport activity compared to control transformants  (transformed with vector pBinAR without insertion), as well as the C / N ver ratio, photosynthesis rate, photorespiration and growth.

Claims (19)

1. DNA sequences which contain the coding region of a glutamate / malate translocator, characterized in that these DNA sequences have the nucleotide sequences shown in FIG. 1 (Seq ID No .: 1-3). 2. DNA sequences with the DNA sequences according to claim 1 or parts thereof or derivatives derived from insertion, deletion or substitution of these Sequences are derived, hybridize and code for a plastid protein, which has the biological activity of a glutamate / malate translocator. 3. Plasmids containing DNA sequences according to claim 1 or 2. 4. Phagemid pSbGMT1 deposited under the DSM number DSM 12480 as E. coli Strain DH5α pSbGMT1 containing this plasmid. 5. Phagemid pFbGMT1 deposited under the DSM number DSM 12478 as E. coli Strain DH5α pFbGMT1 containing this plasmid. 6. Phagemid pSoGMT1 deposited under the DSM number DSM 12479 as E. coli Strain DH5α pSoGMT1 containing this plasmid. 7. Bacteria containing a DNA sequence according to claim 1 or 2 or containing phagemids according to claims 3 to 6. 8. yeast containing a DNA sequence according to claim 1 or 2 or Plasmids according to claim 3. 9. Bacteria containing a DNA sequence according to claim 1 or 2 or containing plasmids / phagemids according to claims 3 to 6.   10. plant cells containing plasmids according to claims 1 and 2. 11. Transformed plant cells and transgenic plants regenerated therefrom containing DNA sequences according to claim 1 or 2, wherein these sequences as Part of a recombinant DNA molecule introduced into the plant cells were. 12. Use of DNA sequences according to claim 1 or 2 to Introduction to pro- or eukaryotic cells, these sequences with Controls that ensure transcription and translation in the cells, are linked and for the expression of a translatable mRNA which supports the synthesis of a glutamate / malate translocator. 13. Use of a DNA sequence according to claim 1 or 2 for the isolation of DNA sequences that code for a polypeptide that expresses the biological activity of a Has glutamate / malate translocators. 14. Use of a DNA sequence according to claim 1 or 2, being this sequence contains a coding region that is responsible for a mature protein with the biological Coded activity of a glutamate / malate translocator for combination with "Targeting" sequences for other cell compartments or cellular Membrane systems, whereby the mature protein in other compartments or Membrane systems is conducted. 15. Use of a DNA sequence according to claim 1 or 2 to identify Substances that have an inhibitory effect on the transport of dicarboxylic acids over the inner plastid envelope membrane. 16. Use of DNA sequences according to claim 1 or 2 to Isolation of corresponding genomic clones. 17. Use of promoter areas or of partial promoter areas according to Claim 16 for tissue-specific expression of genes.   18. Use of DNA sequences according to claim 1 or 2 to Production of DNA sequences for a plant plastid Code the dicarboxylic acid translocator with a changed specificity. 19. Use of DNA sequences according to claim 1 or 2 to Identification of insertion mutants, for homologous recombination or for Expression of a non-translatable RNA, which by means of an "antisense" effect, a cosupression or a ribozyme activity the synthesis of one or more prevents endogenous plastid glutamate / malate translocators in the cells.