DE19826444C1 - DNA encoding a spinach glucose translocator, plasmids, bacteria, yeast and transformed plant cells - Google Patents

Abstract

A DNA sequence (I) containing the coding region of a glucose translocator that is characterized by a 1864 bp sequence (given in the specification) is new. Independent claims are also included for: (1) a plasmid containing a DNA sequence which is derived through insertion, deletion or substitution from (I); (2) bacteria or yeast containing (I), in particular a plasmid or phagemid as above; (3) a plant cell containing a plasmid as above; and (4) a transformed plant cell and regenerated transgenic plants from this containing an introduced recombinant (I).

Description

The present invention relates to a DNA sequence from Spinacia oleracea (spinach), which contains the coding region of a glucose translocator, its introduction into a plant genome the formation and transfer of carbon skeletons in transgenic plants altered, containing plasmids, yeasts and bacteria DNA sequences, as well as transgenic plants in which the introduction of DNA Sequence changes in the activity of the glucose translocator and thus Changes in carbon change are caused. Furthermore concerns the invention the use of the described sequence of the glucose translocator to identify related translocators from other plants (Angiosperms, gymnosperms, as well as algae) through hybridization with lower Stringency or by PCR techniques, as well as the use of the glucose translocator as a target for herbicides.

Plant photosynthesis is the largest production process on earth and the only process that increases the total supply of usable energy on our planet: more than 10 billion tons of carbon are stored annually in carbohydrates or other organic matter as energy and ultimately as our food base. This amount of energy corresponds to about 5% of all known fossil energy sources. Photosynthesis is the basis of all life. During this process, which takes place in photosynthetically active plastids, adenosine triphosphate (ATP) and reduction equivalents (NADPH ₂ ), a carbon skeleton with 3 carbon atoms, triosephosphates are formed from the atmospheric CO ₂ , inorganic phosphate and water with the help of the products formed in the course of the light reaction. synthesized. This primary product of the CO ₂ fixation is via a special translocator (triose phosphate / phosphate translocator, TPT; Flügge et al., 1989, EMBO J. 8: 39-46; Flügge et al., 1991, Nature 353: 364-367 ) transported out of the chloroplast and converted into sucrose (cane sugar) in the cytoplasm in a series of successive reactions. During the biosynthesis of sucrose, the inorganic phosphate is released again and transported back into the chloroplasts via the triose phosphate / phosphate translocator mentioned above. Here ATP is again formed from the phosphate, a reaction that is catalyzed by the light-driven ATP synthase. It can be seen that the export of the carbon fixed during the day, catalyzed by the triose phosphate / phosphate translocator, in an exchange with inorganic phosphate, essentially connects the two compartments chloroplast and cytoplasm.

During the light period, the triose phosphates formed by photosynthesis can also be converted into starch, the so-called assimilation starch. This process takes place in the chloroplasts and serves to build up a transient carbon store that the plant can use during the subsequent night period. Furthermore, carbon can also be assimilated in this way if the sucrose biosynthesis in the cytosol does not keep pace with the net CO ₂ assimilation. In the following dark period, the starch storage is broken down again and the fission products of the starch mobilization are removed from the chloroplasts and, after further conversion into the form of transport of the photoassimilate (primarily sucrose), exported from the leaf. This ensures that the non-green tissues are continuously supplied with photoassimilates, both during the day and during the night.

The mobilization of the assimilation strength can be done both through a phosphorolytic as well as hydrolytic degradation. In the first Fall comes to hexose phosphates and ultimately to triose phosphates that over the above-mentioned TPT can be exported from the chloroplasts. in the The cytosol is then converted further to sucrose. First, under Catalysis of the triose phosphate isomerase and the aldolase from the triose phosphates Glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate fructose-1,6- bisphosphate formed. These reversible equilibrium reactions are caused by the Cleavage of a phosphate group from fructose-1,6-bisphosphate in the direction Fructose-6-phosphate advanced. However, this continues the activity of the cytosolic fructose-1,6-bisphosphatase (FBPase), which is considered one of the Key enzymes for sucrose biosynthesis can be viewed. The FBPase, however, is under the strict control of fructose 2,6-bisphosphate, one efficient negative modulator. The concentration of this FBPase inhibitor shows has a diurnal rhythm, is low in the light and so high in the dark that here the FBPase is largely switched off (Stift, 1990, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 153-185). If the FBPase is not active, the plastids can exported triose phosphates cannot be converted into sucrose. That also says the exchange substrate required for the export of triose phosphates Phosphate not available. In the plastid, however, phosphate would serve as a substrate for the phosphorolytic starch degradation needed. It can therefore be assumed that the Nocturnal starch mobilization primarily via hydrolytic degradation he follows. Experiments with a high-strength mutant, TC265, also confirm this Guess (see below). As a limitation, it must be noted here that it There may be plant species in which the starch breakdown is predominant  takes place phosphorolytically (e.g. Erbsen, Stitt et al., 1978, Biochim. Biophys. Acta 544: 200-214).

In contrast to the phosphorolytic degradation of the starch, the hydrolytic provides Starch breakdown - via amylases, maltases etc. - glucose, the basic building block of Strength. Glucose is transported through a special transporter, the glucose translocator exported from the chloroplast. After phosphorylation to glucose-6-phosphate through the enzyme hexokinase can glucose-6-phosphate through the Glucose phosphate isomerase can be converted into fructose-6-phosphate. This Reaction sequence bypasses the FBPase reaction. Another molecule of glucose-6 Phosphate is rearranged to glucose-1-phosphate by phosphoglucose isomerase and then from UDP-glucose pyrophosphorylase (UGPase, glucose-1-phosphate- Uridylyltransferase) converted to UDP-glucose. UDP-glucose and fructose-6 phosphate are then derived from the sucrose phosphate synthase and the Sucrose phosphate phosphatase converted to sucrose.

It can thus be stated that during the ongoing photosynthesis the Export of the photoassimilate formed takes place predominantly via the TPT, while At night, however, the glucose translocator played a crucial role in the frame of the export of mobilized photoassimilate. In the dark period the glucose translocator therefore essentially connects the two Compartments of chloroplast and cytoplasm. The crucial role of glucose Translokators in the export of starch mobilization products is through following experiments confirmed:

Recently, transgenic plants with a modulated activity of chloroplastic TPT are produced. The inhibition of this transporter in planta on the expression of a corresponding "anti-sense" mRNA leads to a reduced export of the primary photoassimilate (triose phosphate, 3- Phosphoglycerate) which, under these circumstances, enters the chloroplast within the Biosynthesis is derived from starch that accumulates massively (Riesmeier et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6160-6164; Heineke et al., 1994, Planta 193: 174- 180). It could also be shown that in the transgenic plants accumulated starch can in turn be mobilized to a greater extent, whereby this Mobilization depending on the plant species during the night (potato; Heineke et al., 1994, Planta 193: 174-180) or during the day (tobacco, Hausler et al., 1998, Planta 204: 366-376). Bypassing the in its activity reduced TPT in the transgenic plants, the starch is mobilized the hydrolytic route and the export of the released glucose via the Glucose translocator. It could also be shown that in the transgenic anti- sense TPT plants the activity of α-amylase, hexokinase and glucose Translocator is 2-3 times higher than in the control plants.  

The assumption that the products of the starch mobilization of the chloroplast mainly left as glucose is also left by the biochemical Characterization of a so-called "starch excess" Arabidopsis mutant, TC26, supported, which has a constantly high content of starch (Caspar et al., 1991, Plant Physiol. 95: 1181-1188; Trethewey and ap Rees (1994) Biochem. J. 301, 449-454). In principle, this mutant is capable of starch due to its enzyme content break down, however, the products of starch breakdown can not from the Export chloroplasts. It could be shown that the TC26 mutant has a functional TPT but does not get glucose from the chloroplasts into it Can transport cytosol. The lack of the glucose translocator leads to one Accumulation of starch breakdown products and thus a new synthesis of starch combined with the observed high starch content. Taken together the experiments discussed underline the crucial importance of the Glucose translocators in the export of carbon skeletons from the Starch breakdown. The glucose translocator was first described in 1977 (Schäfer et al., 1977, Plant Physiol 60: 286-289), a further biochemical or however, molecular characterization of this transport system is pending. The process described and taking place in the leaves (the "source" tissue) photoassimilate production serves to a large extent to supply non- green organs (the "sink" tissue), such as root, tuber and fruit, also the harvestable parts of the plants. In crops such as potatoes, sugar beets and grain, carbohydrates are transported from the "source" to the "Sink" tissues over the sieve tubes (the phloem) exclusively in the form of Sucrose. The sucrose is then extracted from the phloem into the cells of the "Sink" tissue transported (phloem discharge). Transport can either directly d. H. symplastic over between the phloem and the "sink" tissue existing connections (plasmodesms) take place or apoplastically what a discharge of the sucrose into the cell wall and its re-import into the Includes target cells. Alternatively, the sucrose in the apoplast from one cell wall-specific invertase are split into hexoses, which are then the cells of the "sink" tissue are transported. Is it transported to Form of sucrose, this is in the cytoplasm of the cells of the "sink" tissue in Split hexoses (glucose and fructose). This is done either via invertases or via sucrose synthase. All sucrose cleavage products are ultimately again in hexose phosphates (glucose-6-phosphate, glucose-1-phosphate) transferred.

In most plants, glucose-6-phosphate then serves as a substrate for the Starch synthesis, the entire reaction sequence in the interior (stroma) of Amyloplast of the "sink" tissue is localized. These organelles are of two  Enclosed lipid bilayer membranes, one of which is the outer membrane Because of the presence of pore-forming proteins (porins) unspecific permeable is for smaller molecules. Such a porin of the outer amyloplast Envelope membrane was recently identified by us and the sequence of its that Protein-composing amino acid building blocks can be elucidated (Fischer et al., 1994, J. Biol. Chem., 269: 25754-25760). The inner envelope membrane, however, is that actual permeability limit between the cytoplasm and the organelles and is, like a priori any lipid bilayer, in principle impermeable to larger ones charged molecules such as B. hexose phosphate. This impermeability in principle the inner amyloplast envelope membrane for larger hydrophilic molecules bridged by the presence of specific transport systems Glucose-6-phosphate is released via a transporter we recently characterized, imported the glucose-6-phosphate / phosphate translocator (GPT) into the plastids (Kammerer et al. (1998) Plant Cell 10: 105-117). This can happen within the plastid imported glucose-6-phosphate was then fed into the starch biosynthesis become. The transport into the amyloplasts takes place in an exchange Mechanism, here in exchange with inorganic phosphate, which in the course of Starch synthesis released. Glucose-6-phosphate is converted into glucose-1-phosphate implemented, which then via ADP-glucose pyrophosphorylase (AGPase) in ADP- Glucose, the substrate for the starch synthase, is implemented. Recent works have shown that in the endosperm of some cereals (barley, maize) the AGPase too is localized in the cytosol and the introduction of the precursors for the Starch biosynthesis can take place via ADP-glucose (Denyer et al., 1996, Plant Physiol. 112: 779-785).

An exception to this may be fat-storing castor plastids communis. Here there is evidence that glucose is obtained directly from a glucose Transporter is taken up from the cytosol into the plastids and through there a plastid hexokinase is phosphorylated to glucose-6-phosphate. (Dennis and Miernyk, 1982, Ann. Rev. Plant Physiol. 33: 27-50).

The imported glucose-6-phosphate is also the starting substrate for the oxidative Pentosephosphate route (OPPP), the reduction equivalent, mainly for the reduction of the nitrite formed from the nitrate to Ammonium, which in turn is involved in the biosynthesis of amino acids / proteins is dissipated, provides. In this case, glucose-6 is transported phosphate in exchange with triose phosphates, the products of the OPPP (Kammerer et al. (1998) Plant Cell 10: 105-117).

The starch formed in the non-green plastids is subject to, as is that Assimilation strength of chloroplasts, a permanent build-up and breakdown (stitt and Heldt 1991, Biochim. Biophys. Acta 638: 1-11). Is the rate of synthesis  higher than that of mining, the strength is considered a long-term store for that fixed carbon. The strength stored in the (harvestable) stores then serves as the basis for animal / human nutrition (e.g. starch in Potato tubers, corn kernels). When seeds germinate, the starch becomes red mobilized and the starch breakdown products then serve as fuel for the emerging seedling. It can be assumed that, as shown above using the example of the Degradation of chloroplastic assimilation starch, the glucose Translocator of the non-green plastids in the mobilization of the storage strength play a crucial role.

As can be seen from the explanations, transporters are like that chloroplastic triosephosphate-phosphate translocator, which prevents the transport of the fixed carbon catalyzed from the chloroplast, the sucrose Translocator, over which the sieve tubes with the transport form of the photoassimilate Sucrose are loaded, the glucose-6-phosphate-phosphate translocator Amyloplast, which provides the starting substrate for the starch synthesis, or the Glucose translocator, via which the breakdown products of starch mobilization Plastids leave - in the allocation of the photoassimilate from the "source" to the "Sink" organs integrated at a crucial point. 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 Triosephosphate-phosphate translocator can be influenced (see above). Also the reduction of sucrose transport activity via an "anti-sense" inhibition of the sucrose transporter responsible for transport into the sieve tubes (Riesmeier et al., 1992, EMBO J. 11: 4705-4713) leads to a drastic phenotype of plants: they are much smaller, the leaves are damaged and the plants in the case of potatoes, hardly form tubers from d. H. the supply of the "Sink" tissue with photoassimilates is greatly reduced (Riesmeier et al., 1994, EMBO J. 13: 1-7).

As stated, the glucose translocator plays a central role in that Transport of degradation products of starch hydrolysis from photosynthetically active and non-green plastids too. If this translocator could be cloned and with With the help of the sequence obtained, the activity of the translocator in plants change, this opened z. B. the possibility of the starch content of the plants and thereby increasing their dry matter.

As Stark et al. (Science 258: 287-292) 1992, the river was unable to Direction of starch synthesis in potato tubers via the overexpression of an ADP Glucose pyrophosphorylase (AGPase), an amyloplastid enzyme of starch-  Biosynthesis. An enzyme from E. coli was used for these experiments used that does not control metabolic control of the 3- Phosphoglycerate / phosphate quotient is subject to more detailed analysis of the However, transgenic plants showed that the increased carbon flow in the Strength was associated with an increased strength turnover due to one increased starch degradation (Sweetlove et al., 1996 Biochem. J. 320: 493-498).

It can be concluded that, in order to achieve an increased starch content in plants, preferably the turnover, d. H. the starch breakdown and further use and the export of the degradation product glucose must be prevented. Plants with a reduced activity of the plastid glucose translocator could Products of amylolytic starch breakdown, glucose, no longer from the plastids export. Thus, the glucose would immediately build up in starch again introduced or the further starch degradation inhibited by its product glucose and the starch content can be increased. This can also be done derive from the studies on the high-strength mutant described above TC26, which is high in content due to deficiency in the glucose translocator Has strength.

An increase in the starch content of the potato tuber would be for both starch processing industry as well as for the food industry of Importance since potatoes have an increased starch content during their Processing to chips, french fries, pastries etc. absorb less fat and therefore lead to lower calorie end products. Successful strategies would be with high probability also on the use of other starch-storing Plants such as B. corn, wheat and barley transferable.

Potatoes with reduced activity of the glucose translocator would succeed could also generate storage losses caused by so-called "cold-sweetening" arise, be avoided. To avoid germination, potatoes are stored at low temperatures (6-8 ° C). However, this leads to the undesirable Effect of the accumulation of soluble sugars, the "cold sweetening" mentioned. The formation of reducing sugars is, due to the Maillard Response, an extremely negative parameter during the further processing of the Potatoes like making chips etc. The formation of soluble sugar - Hydrolysis products of starch that accumulate in the extraplastid compartment - could be prevented by the glucose translocator that is in the Transport of the products from the starch breakdown is integrated, could be switched off during the storage phase of the potatoes, strengthened this effect could be achieved by a simultaneous reduction of the one we have recently described glucose-6-phosphate / phosphate translocator, which is derived from the  phosphorolytic starch degradation derived from the phosphorylated C3 body Plastids exported (Kammerer et al., 1998, Plant Cell 10: 105-117).

As explained above, the photoassimilates formed in the course of the photosynthesis are mainly exported via the TPT during the day. Experiments with transgenic plants, in which the activity of the TPT was either reduced or increased by repression or overexpression of the corresponding RNA, show that the TPT can have a limiting effect on photosynthesis and the biosynthesis of sucrose, especially under high light and with optimal CO ₂ supply (Häusler et al., 1998, Planta 204: 366-376; Häusler et al., Unpublished observations). It is conceivable to achieve an increased derivation of the fixed carbon via an overexpression of the glucose translocator, especially with a simultaneous overexpression of the ADP-glucose pyrophosphorylase, which leads to an increased starch turnover due to an increased starch breakdown (sweetlove et al., 1996 Biochem. J. 320: 493-498). The fixed carbon would alternatively be provided here as glucose, which is discharged to the chloroplast via the glucose translocator.

The addressing of the plastid translocators to the inner envelope membrane of the Plastids require a corresponding presequence ("targeting sequence") that the attached mature protein correctly directed to the plastids (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; Flügge, 1990, J. Cell Sci. 96: 351-354). Next the pre-sequence necessary for "plastid addressing" is in the mature part (the Part of the protein obtained after the presequence has been cleaved by a specific Protease remains) of the plastid membrane proteins still more information, which are responsible for the specific insertion of the proteins into the membrane and a transport of the envelope membrane proteins across the envelope membrane into the Prevent plastid stroma or, in the case of chloroplasts, the thylakoid membrane (Knight and Gray, 1995, Plant Cell 7: 1421-1432 or own investigations: Brink et al., 1995, J. Biol. Chem. 270: 20808-20815). Own work also showed on Example of a mitochondrial carrier, the ADP / ATP transporter, that this Protein not or only with a very low efficiency directed to plastids and can be installed there in the envelope membrane. Even a hybrid protein consisting of a plastid pre-sequence (the information for the plastid Containing addressing) and this mitochondrial carrier, showed opposite the authentic protein a hardly increased incorporation into the plastid Envelope 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 procaryotic transporters functionally into the inner plastid envelope membrane integrate. With the current state of the art, it is more promising that DNA sequence necessary for the construction of the plants described above by cloning the authentic plastid glucose translocator.

Although this transport system catalyzes the transport of glucose, it is Primary structure is believed to be very different from that of glucose Transporter from the plasma membrane. These vans are part of a large one Gene family of the monosaccharide transporters (Sauer and Tanner, 1993, bot. Acta 106: 277-286; Weig et al., 1994, J. Plant Physiol. 143: 178-183).

To determine the primary structure of the amyloplastid glucose translocator, therefore had to take the extremely difficult route of membrane proteins biochemical characterization, purification and isolation of the transport protein be followed. Starting from chloroplastid shell membranes made of spinach succeeded in combining this protein with chromatographic methods Substrate protection experiments versus labeling with a radioactive labeled inhibitor N-ethylmaleimide - as a component of the inner Chloroplast envelope membrane with an apparent molecular mass of approx. Identify 43,000 daltons.

The N-terminus of the translocator proved to be chemically modified and thus N-terminal protein sequencing through automated Edman degradation inaccessible. It was therefore necessary to prepare the protein by preparative SDS Isolate polyacrylamide gel electrophoresis in sufficient quantity and with a Cleave protease (Lys-C) enzymatically to determine internal peptide sequences (see embodiment). In this way it could do the following Peptide sequence obtained: (1) KGRSLEEIELALSPAV. The 3'- RACE method (rapid amplification of cDNA ends) and using degenerate (modeled after the peptide obtained) synthetic Oligonucleotides to obtain a specific PCR fragment. This was called a probe for the subsequent screening of a cDNA library from spinach leaves used and succeeded in isolating a cDNA clone that the complete cDNA  (1864 base pairs) for the precursor protein of chloroplastic glucose Contained translocator with a molecular mass of 57.6 kDa (see Embodiment 2).

A comparison of the cDNA sequence obtained with sequences from the databases showed that only slight similarities with the known glucose transporters of the Plasma membrane were present (<30%) and the homologies to bacterial Hexose transporters were in the range of approx. 45%. An approx. 73% homology to a hypothetical sugar transporter from Prunus armeniaca (apricot). The derived molecular mass of this transporter is approx. 49.8 kDa. Obviously this was not due to a sequencing error recognized that this protein has an additional N-terminal extension, the could serve as a plastid addressing signal. An assignment to a cellular membrane or an organelle could therefore be used for that in apricot identified gene cannot be hit. However, we were able to show that the chloroplastic glucose translocator is synthesized as a precursor protein as well post-translational and with cleavage of the N-terminal signal sequence into the Chloroplasts are imported and inserted into the envelope membranes (see Embodiment 3).

The present invention provides a DNA sequence which originates from a plant genome and codes for a plastid glucose translocator, the information contained in the nucleotide sequence leading to the formation and expression of a ribonucleic acid in plant cells upon introduction and expression in plant cells Glucose translocator activity can be introduced into the cells or an endogenous glucose translocator activity can be suppressed. The invention relates in particular to the DNA sequences from Spinacia oleracea (spinach) with the nucleotide sequence Seq-ID No .: 1 ( FIG. 1). The invention further relates to DNA sequences which are identified by the sequence shown in SEQ ID NO. 1 mentioned DNA sequence or parts thereof or derivatives which are derived from these sequences by insertion, deletion or substitution, hybridize and code for a plastid protein which has the biological activity of a glucose translocator.

In addition, the present invention relates to the use of the DNA sequence according to the invention or parts thereof or derivatives which by Insertion, deletion or substitution are derived from this sequence for Transformation of pro- and eukaryotic cells. To the expression of the glucose To ensure translocators in transformed cells DNA sequence according to the invention are introduced in vectors and thereby Controls for expression in prokaryotic or eukaryotic  Cells can be combined (see exemplary embodiments 3, 5). Such Control elements are, on the one hand, transcription promoters and, on the other hand Transcription terminators. With the vectors, eukaryotic cells can are transformed with the aim of expressing a translatable Messenger ribonucleic acid (RNA), which is the synthesis of a plastid glucose Translocators allowed in the transformed cells, or with the aim of Expression of a non-translatable, inversely oriented ("anti-sense"), Messenger ribonucleic acid, which is the synthesis of the endogenous glucose translocator prevented. For this purpose, shorter fragments of the DNA sequence according to the invention with a relatively high degree of homology (more than approx. 65% homology) can be used. A repression can also go over the overexpression of the homologous DNA can be achieved (co-suppression). As well can express endogenous glucose translocators through expression a ribozyme constructed for this purpose using the DNA sequences according to the invention are inhibited; also about a Insertion mutagenesis - or as soon as the appropriate technology is available stands for a homologous recombination ("knock-out" mutant). By a tissue-specific controlled expression of an RNA corresponding to the Sequence of a plant glucose translocator according to the invention is a Modification of plant carbon metabolism possible economic importance is that an improved derivation of the Photoassimilate is achieved from the chloroplasts or the degradation products of the Starch mobilization can be retained in the plastids.

Plants can therefore be produced which have an increased starch content have and / or - in the case of z. B. the potato - reduced storage losses exhibit. This modification increases the nutritional value of Plants and thus their economic value. The respective effects left may increase if the tissue-specific expression of the Glucose translocators in the leaf or in heterotrophic "sink" tissue with the further translocators / enzymes of carbon / nitrogen metabolism would be combined (simultaneous expression of multiple target proteins such as the Increasing the starch content or reducing the storage losses of the Potato).

Furthermore, the heterologous expression of the sequence described in Split yeasts (embodiment 4 and Loddenkötter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2155-2159) Structure-function studies of the glucose translocator, the ultimately to develop a specific inhibitor for this protein could lead; Herbicide development is particularly important here, as the Inhibition of a protein with a key function in the metabolic process  would be inevitably lethal to the plant. Furthermore, these are structural-functional studies Basis for a directed mutagenesis of the substrate binding site of the Translocators to specifically change the substrate specificity of the transporter. There are already processes for the genetic modification of dicotyledons and monocotyledons Plants known (Gasser and Fraley, 1989, Science 244: 1293-1299; Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Mol. Biol. 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). Of 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 synthetically produced or naturally obtained or one Contain a mixture of synthetic and natural DNA components. For Preparing for the introduction of foreign genes into higher plants is a big one Number of cloning vectors available that provide a replication signal for E. coli and include a marker that allows selection of the transformed cells. Examples of vectors are pBR322, pUC series, M13 mp series, pACYC 184 etc. After inserting desired genes into the plant, additional DNA Sequences may be required. Are z. B. for the transformation of the plant the Ti or Ri plasmid used, at least the right boundary, often however, the right and left boundaries of the Ti and Ri plasmid T-DNA as Flank area to be added to the genes to be introduced. The use of T-DNA for the transformation of plant cells has been studied intensively and sufficiently described (Hoekema, in: The Binary Plant Vector System, Offset drukkerij 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 introduced DNA once in the Genome integrated, so it is usually stable there and remains in the Progeny of the originally transformed cell obtained. It contains usually a selection marker that unites the transformed plant cells Resistance to biocides (like Basta) or antibiotics (like Kanamycin, Bleomycin or hygromycin) gives. The individually introduced marker will hence the selection of transformed cells over cells to which the introduced DNA is missing, allow.

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 sequence 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 glucose translocator can be changed e.g. B. towards one Affinity for fructose or galactose.

Likewise, an insensitivity of the glucose translocator could be specific Herbicides can be achieved. Using 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 and / or synthetic or natural sequences are added. For the Connection of the DNA fragments to one another can be made using adapters or links can be scheduled. 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 yeast and measurement of the modified Transport properties in artificial liposomes (see embodiment 4; Loddenkötter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2155-2159; 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 recently developed protein for this purpose Method (Flügge and Weber, 1994, Planta, 194: 181-185) applied.

The DNA sequence according to the invention (or parts or derivatives of these sequences) can be used according to standard procedures (in particular Hybridization screening of low stringency cDNA libraries using the DNA sequence according to the invention or parts of the DNA  Sequence as a probe or creating probes for stringent and low stringent screening strategies by deriving degenerate ones and / or non-degenerate primers from the DNA sequence according to the invention for PCR experiments with DNA or cDNA from other plants) from the genome isolate from plant-like sequences that code for proteins that also transport glucose.

The DNA sequence according to the invention (Seq. ID No. 1) contains protein in the translated Areas capable of being synthesized on ribosomes in the cytoplasm To direct protein specifically to plastids and the appearance of the protein in to prevent other membrane systems of the cell. The protein area that does that directs protein encoded by the DNA sequence according to the invention to plastids, lies within the first 90 amino acids of the protein, is for the transport function of the protein is not required and will be inserted into the protein after successful insertion the plastid covering membrane is removed. By exchanging this "plastid targeting" - Sequence against one of the known "targeting" sequences e.g. B. for mitochondria the translocator protein could be more eukaryotic in another membrane system Conduct cells and could possibly use the transport properties there change the respective membrane. Likewise, the "plastid targeting" sequence could of the glucose translocator or endogenous regions of the mature protein foreign proteins (e.g. bacterial transport proteins or transporters Yeast) into the plastids or into the plastid envelope membrane of plant cells conduct.  

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

1. Cloning procedure

The phage LambdaZAP II and the phagemid pBluescript were used for cloning II SK (pBSC) (Short et al., 1988, Nucl. Acids Res. 16: 7583-7600).

For the transformation of yeast, the vector pEVP11 (Russel and Nurse, 1986, Cell 45: 145-153).

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 pBluescriptSK (pBSC) phagemid as well as for pEVP11 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 was analyzed by electrophoresis for correctness and orientation.  

4. Plant transformation

15 small leaves of a sterile tobacco culture were transformed per transformation Conditions with a scalpel in pieces of about 1 cm edge length cut up. The leaf pieces were transferred to MS medium with 2% glucose, in which then with an inoculation loop from one under strict selection Transformed Agrobacterium tumefaciens culture grown on agar plates Bacterial cells were stirred into the medium. After 30 minutes of incubation the leaves were on MS medium with 1.6% glucose for two days in the dark Incubated at room temperature. The leaf pieces were then placed on an MS Medium with 2% sucrose, 2 mg / l kinetin, 500 mg / l Betabactyl®, 15 mg / l Hygromycin (or 100 mg / l kanamycin) and 0.8% Bacto agar designed. After one week incubation at 25 ° C and 3,000 lux illuminance Beta-lactyl concentration in the medium reduced by half. From the Leaf pieces grew without pronounced callus phase, small shoots, which for Root formation on a MS medium with 2% sucrose, 250 mg / l betabactyl and 100 mg / l kanamycin were transferred. After root formation, the Saplings transformed into earth culture.

Arabidopsis was transformed using the method of Bechtold et al., 1993, C.R. Acad. Sci. 316: 1194-1199.

Filing:

On June 10, 1998 the following phagemid in the form of an E. coli strain containing this phagemid was deposited with the German Collection of Microorganisms (DSM) in Braunschweig, Federal Republic of Germany:

Phagemid: pBSC-E43 / 30-3 (DSM 12243)

Embodiment 1 Isolation of the glucose translocator, extraction of peptide fragments of this translocator and creation of probes for hybridization screening of cDNA libraries.

Identification of the glucose translocator in the envelope membranes of spinach Chloroplasts were made using a combination of radioactive labeling Experiments and substrate protection experiments. It could be shown that the Transport of glucose into isolated chloroplasts by very low Concentrations of the sulfhydryl reagents pCMBS and NEM can be inhibited could.  

Isolated chloroplasts were in the presence or absence of glucose, Maltose and sorbitol with the radioactively labeled sulfhydryl reagent N- Ethylmaleimide (NEM) added. Then the chloroplast Envelope membranes isolated, separated by SDS-PAGE and separated from the fixed stained and dried gels fluorographs made. It turned out that a Protein with an apparent molecular mass of 43 kDa in SDS-PAGE below differentially marked under the different conditions. It could continue can be shown that this protein is missing in the Arabidopsis mutant TC26 and not is marked.

The protein identified in this way as a glucose translocator was then from the chloroplast envelope membranes by extracting the Membranes with a mixture of chloroform and methanol (2: 1) together with extracted four more proteins. The high lipid extract was one Separation in preparative SDS gels was not directly accessible but had to be are first delipidated with hexane.

The protein was then prepared in preparative SDS-polyacrylamide gels (Laemmli, 1970, Nature 227: 680-685). After detection of the protein by staining with Coomassie-Brilliant Blue R-250 a 43 kDa protein band was made from the gel cut out and cleaved in the gel matrix with the endoprotease Lys-C (Fresco, 1979, Anal. Biochem. 97: 382-386). The resulting peptides were made from the gel eluted and separated by HPLC (Eckerskorn and Lottspeicher, 1989, Chromatographia, 28: 92-94). The amino acid sequence of the purified Peptide fractions were automated by Edman gas phase degradation determined (Eckerskorn et al., 1988, Eur. J. Biochem. 176: 509-519). From the Amino acid sequence of one of the two peptides (peptide 1) were two degenerate oligonucleotide sequences encoding this amino acid sequence, derived and the oligonucleotide in question by in vitro DNA synthesis manufactured.

Embodiment 2 Cloning of the glucose translocator from spinach leaves

Spinach leaves were harvested in the middle of the light period and in the middle of the dark period and stored at -80 ° C until RNA isolation. From this, polyA ⁺ RNA was isolated and from this a cDNA library was created in the vector LambdaZAPII.

On the other hand, the isolated RNA was used for an RT polymerase chain reaction (PCR) (RACE; Schaefer, B.C. (1995) Anal. Biochem. 227, 255-273). As a primer for the first RACE reaction was the first specific oligonucleotide in Combination with an "anchor (dT) 15" primer used. The products received  were subjected to a second PCR, with the respective "nested" primers were used. A specific PCR fragment (300 bp) could be obtained become.

Approx. 300,000 clones from the cDNA library were probed with this PCR fragment (see embodiment 1). Clones that reacted positively were recreated Standard procedures cleaned and after preparation of the amplified phage DNA the inserted plaques became EcoRI restriction digestion obtained coding for the glucose translocator and by Southern blot analysis, with the above-mentioned PCR fragment as a probe. After in vivo excision of the phagemid from the phage with the help of the filamentous helper phage ExAssist the clones were analyzed by determining the DNA sequence (Dideoxy method: Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) and the primary structure of the glucose translocator is derived from this DNA sequence (Clone pBSC-E43-30 / 3). The sequence of the peptide could be in the overall sequence of the Translocators can be found.

Embodiment 3 Addressing the glucose translocator precursor protein to the chloroplasts and energy-dependent insertion of the mature protein into the inner membrane.

The in vitro transcription of the plasmid pBSC-E43-30 / 3 was used of T7 RNA polymerase according to the manufacturer's instructions (Pharmacia) carried out. The subsequent in vitro translation was carried out in the reticulocyte lysate (Boehringer-Mannheim) and the postribosomal supernatant was used for Protein transport used in intact spinach chloroplasts. The experiment was carried out in Performed in the dark or in the light; the approach contained import buffers (Flügge et al., 1989, EMBO J. 8: 39-46) and intact spinach chloroplasts (corresponding to 200 µg Chlorophyll). After 30 min at 25 ° C the chloroplasts were washed and the Envelope membranes isolated (Flügge et al., 1989, EMBO J. 8: 39-46). You were over one SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227: 680-685) and subsequent fluorography (Bonner and Laskey, 1974, Eur. J. Biochem. 46: 84-88) analyzed. The presequence of the glucose translocator was found to be the attached mature protein correctly to its target membrane, the inner chloroplast Envelope membrane conducts; it will be replaced by a cleave specific protease and the mature protein is formed. Could in the dark the insertion of the translocator can be energized by the addition of ATP while no import was observed in the absence of ATP. In the light could the energy for the import of the protein in the form of ATP over the photosynthetic phosphorylation are provided; the import was under these conditions regardless of added ATP. Was the  Photosynthetic phosphorylation and the related production of However, ATP was prevented by adding a decoupler like CCCP Protein import blocked. It was also shown that the mature protein is proteae resistant was inserted into the inner membrane: addition of proteases (e.g. Thermolysin), which cannot permeate the outer envelope membrane, can do this not attack mature protein built into the inner membrane. A Pretreatment of the chloroplasts with a protease (e.g. thermolysin) resulted in a total loss of binding and import of the translocator which showed that the pre-sequence ("targeting" sequence) of the translocator in a first step must first be specifically bound to receptors of the outer membrane. Only then can the further steps of protein insertion take place.

Embodiment 4 Expression of the glucose translocator from spinach in the split yeast Schizosaccharomyces pombe

For the expression of the glucose translocator in yeast, PCR was first used an oligonucleotide that is at the intersection of pre-sequence and mature Protein is, the corresponding fragment produced. The fragment thus obtained was inserted into the yeast expression vector pEVP11 and after amplification of the construct in E. coli in LiCl / PEG made competent (Ito et al., 1983, J. Bact. 153: 163-168) Leucine synthesis deficient S. pombe cells transformed (Fischer et al., 1997, Plant Cell, 9: 453-462; (Kammerer et al., 1998, Plant Cell 10: 105-117) Transformants were selected by selection on minimal medium without leucine selected because the pEVP11-E43-30 / 3 construct gives the yeast cells the ability to Gives growth on leucine-free medium.

Embodiment 5 Transformation of plants with a construction for overexpression of the coding region of the glucose translocator

From the phagemid pBluescript-E43-30 / 3 (pBSC-E43-30 / 3), which as the insertion Contained cDNA for the spinach glucose translocator (see Embodiment 2) the insertion was isolated by restriction digestion and in directed the vector pBinAR (Höfgen and Willmitzer, 1990, Plant Sci. 66: 221-230) cloned. After amplification of the resulting construct pBinAR-E43-30 / 3 in E. coli the construct was transformed into agrobacteria and these were then transformed into Infection of leaf segments from tobacco and potato are used. The transformants obtained were analyzed using Southern blot analyzes investigated the presence of the intact, non-rearranged chimeric gene. The  Glucose transport activity was compared to control transformants (transformed with vector pBinAR without insertion), as well as the C / N Ratio, photosynthesis rate, starch content and growth.

Claims (17)

1. DNA sequence which contains the coding region of a glucose translocator, characterized in that this DNA sequence has the nucleotide sequence shown in FIG. 1 (Seq-ID No .: 1). 2. DNA sequence with the DNA sequence according to claim 1, or parts thereof or derivatives derived from insertion, deletion or substitution of this Sequence is derived, hybridize and encode a plastid protein that the has biological activity of a glucose translocator. 3. Plasmids containing a DNA sequence according to claim 1 or 2, which by Insertion, deletion or substitution is derived from this sequence. 4. Phagemid pBSC-E43 / 30-3 filed under the DSM number DSM 12243 as E. coli strain DH5α pBSC-E43 / 30-3 containing this phagemid. 5. Bacteria containing a DNA sequence according to claim 1 or 2 or containing plasmids / phagemids according to claims 3 or 4, which by Insertion, deletion or substitution is derived from this sequence. 6. yeast containing a DNA sequence according to claim 1 or 2 or Plasmids according to claim 3, which are obtained by insertion, deletion or substitution of this sequence is derived. 7. plant cells containing plasmids according to claims 1 and 2. 8. 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 has been. 9. Use of DNA sequences according to claim 1 or 2, by insertion, Deletion or substitution derived from these sequences for introduction to Pro or eukaryotic cells, these sequences possibly with Controls that ensure transcription and translation in the cells, are linked and for the expression of a translatable mRNA, which is the synthesis of a glucose translocator.   10. Use of DNA sequences according to claim 1 or 2, which by Insertion, deletion or substitution are derived from these sequences for Identification of insertion mutants, for homologous recombination or for Expression of a non-translatable RNA, which by means of an "anti-sense" effect, a cosupression or a ribozyme activity the synthesis of one or more prevents endogenous plastid glucose translocators in the cells. 11. Use of a DNA sequence according to claim 1 or 2, by insertion, Deletion or substitution derived from this sequence to change the Carbon / nitrogen ratio in leaves or in heterotrophic tissues especially to increase the starch content. 12. Use of DNA sequences according to claim 1 or 2, which by Insertion, deletion or substitution are derived from these sequences for Reduced sugar formation during starch mobilization. 13. Use of a DNA sequence according to claim 1 or 2, which by insertion, Deletion or substitution derived from this sequence for the isolation of DNA sequences that code for a polypeptide that expresses the biological activity of a Owns glucose translocators. 14. Use of a DNA sequence according to claim 1 or 2, the "targeting" - Sequence serves to use this sequence to be prokaryotic or eukaryotic Proteins, especially enzymes or proteins that are active or passive Catalyze transport of metabolites across membranes into the To direct the plastid envelope membrane into the plastid stroma or into the thylakoids. 15. Use of a DNA sequence according to claim 1 or 2, by insertion, Deletion or substitution is derived from this sequence, this sequence a coding region contains that for a mature protein with the biological one Coding the activity of a glucose translocator, in combination with "targeting" - Sequences for other cell compartments or cellular membrane systems, whereby the mature protein is directed into other compartments or membrane systems. 16. Use of a DNA sequence according to claim 1 or 2, by insertion, Deletion or substitution derived from this sequence to identify Substances that have an inhibitory effect on the transport of hexoses through the have inner plastid envelope membrane.   17. Use of DNA sequences according to claim 1 or 2, which by Insertion, deletion or substitution are derived from this sequence for Isolation of appropriate genomic clones, especially the use of the corresponding promoter areas or of partial promoter areas for tissue-specific expression of genes.