EP1135499A1 - Dna sequences which code a glucose-translocator, plasmids, bacteria, yeasts and plants containing this translocator - Google Patents

Abstract

The invention relates to DNA sequences from Zea mays (corn), Solanum tuberosum (potato) and Spinacia oleracea (spinach) which contain the coding region of a glucose-translocator. The introduction of said DNA sequences in a vegetable genome modifies the formation and transmission of carbon parent structures in transgenic plants, plasmids, yeasts and bacteria containing these DNA sequences. The invention also relates to transgenic plants in which modifications of the activity of the glucose-translocator and thus alterations in the carbon metabolism are provoked by introducing the DNA sequences. In addition, the invention relates to the use of the described sequences of the glucose translocator for identifying related translocators from other plants (angiosperms, gymnosperms, and algae) by hybridizing with lower stringency or by PCR techniques. The invention also relates to the use of the glucose-translocator as a 'target' for herbicides.

Description

 DESCRIPTION

DNA sequences encoding a glucose translocator, plasmids, bacteria, yeast and plants containing this transporter.

The present invention relates to DNA sequences from Zea mays (maize), Solanum tuberosum (potato) and Spinacia oleracea (spinach), which contain the coding region of a glucose translocator, the introduction of which into a plant genome involves the formation and transfer of carbon frameworks in transgenic plants modified, plasmids, yeasts and bacteria containing these DNA sequences, as well as transgenic plants in which the introduction of the DNA sequences causes changes in the activity of the glucose translocator and thus changes in the carbon metabolism. Furthermore, the invention relates to the use of the described sequences of the glucose translocator for identifying related translocators from other plants (angiosperms, gymnosperms, and algae) by hybridization with low stringency or by PCR techniques, and the use of the glucose translocator as a target (" 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, the atmospheric CO2, inorganic phosphate and water are converted into a carbon skeleton with the help of the products formed in the course of the light reaction, adenosine triphosphate (ATP) and reduction equivalents (NADPH ₂ )

C atoms, triose phosphates, synthesized. This primary product of the C0 ₂ fixation is 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) were transported out of the chloroplast and in the cytoplasm in a number of successive reactions converted into sucrose (cane sugar). 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 access 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 C0 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 starch can take place via a phosphorolytic as well as a hydrolytic degradation. In the first case, hexose phosphates are obtained and ultimately triose phosphates, which can be exported from the chloroplasts using the TPT mentioned above. The further conversion to sucrose then takes place in the cytosol. First, under the catalysis of the triose phosphate isomerase and the aidolase from the triose phosphates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, fructose-1, 6-bisphosphate is formed. These reversible equilibrium reactions are advanced by splitting off a phosphate group from fructose-1, 6-bisphosphate towards fructose-6-phosphate. However, this requires the activity of cytosolic fructose-1,6-bisphosphatase (FBPase), which can be considered one of the key enzymes for sucrose biosynthesis. However, the FBPase is under the strict control of fructose 2,6-bisphosphate, an efficient negative modulator. The concentration of this FBPase inhibitor has a diurnal rhythm, is low in the light and so high in the dark that the FBPase is largely switched off here (Stitt, 1990, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 153- 185). If the FBPase is not active, triose phosphates exported from the plastids cannot be converted into sucrose. The counter-exchange substrate phosphate required for the export of triose phosphates is therefore also not available. In the pasti, however, phosphate would be required as a substrate for the phosphorolytic starch degradation. It can therefore be assumed that the starch is mobilized at night primarily via hydrolytic degradation. A mutant of Flaveria linearis with a defective cytosolic FBPase does not have an obvious phenotype. This mutant shows a reduced carbon export from the leaf during the day, but increased during the night period (Zrenner et al., 1996, Plant J. 9: 671-681). Experiments with a high-strength mutant, TC265, also confirm this assumption (see below). Furthermore, NMR experiments using deuterium-labeled glucose also indicate that during the night most of the carbon leaves the chloroplast as glucose, bypassing the TPT and the cytosolic FBPase (Schleucher et al., 1998, Plant Physiol. 1 18: 1439-1445). As a limitation, it should be noted here that there may be plant species in which the starch degradation takes place predominantly phosphorolytically (for example peas, Stitt et al., 1978, Biochim. Biophys. Acta 544: 200-214).

In contrast to the phosphorolytic degradation of the starch, the hydrolytic starch degradation - via amylases, Maltesen etc. - provides glucose, the basic building block of the starch. Glucose is exported from the chloroplast via a special transporter, the glucose translocator. After phosphorylation to glucose-6-phosphate by the enzyme hexokinase, glucose-6-phosphate can by 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 converted to UDP-glucose by UDP-glucose-pyrophosphorylase (UGPase, glucose-1-phosphate-uridylyl transferase). UDP-glucose and fructose-6-phosphate are then converted to sucrose by the sucrose phosphate synthase and the sucrose phosphate phosphatase.

It can thus be stated that during the ongoing photosynthesis the photoassimilate formed is exported predominantly via the TPT, but during the night the glucose translocator plays a decisive role in the export of mobilized photoassimilate. In the dark period, the glucose translocator therefore essentially connects the two compartments, chloroplast and cytoplasm. The crucial role of the glucose translocator in the export of starch mobilization products is demonstrated by the following experiments:

Recently, transgenic plants with a modulated activity of the chloroplast TPT could be produced. The inhibition of this transporter in planta via the expression of a corresponding "antisense" mRNA leads to a reduced export of the primary photoassimilate (triose phosphate, 3-phosphoglycerate), which under these circumstances is derived within the chloroplast into the biosynthesis of starch which is found massive accumulation (Riesmeier et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6160-6164; Heineke et al., 1994, Planta 193: 174-180). It was also possible to show that the accumulated starch can in turn be mobilized more intensely in the transgenic plants, this mobilization depending on the plant species during the night (potato; Heineke et al., 1994, Planta 193: 174-180) or even during of the day (Tobacco, Häusler et al., 1998, Planta 204: 366-376). Bypassing the TPT, whose activity is reduced, the starch is mobilized in the transgenic plants via the hydrolytic route and the exported glucose is exported via the glucose translocator. It could also be shown that the activity of the α-amylase, the hexokinase and the glucose translocator in the transgenic anti-sense TPT plants is higher by a factor of 2-3 than in the control plants. The assumption that the products of the starch mobilization of the chloroplast leave mainly as glucose is also supported by the biochemical characterization of a so-called "starch excess" / Araό / ops / ^' s mutant (TC26, with a defective sexual gene), which keeps you constant has a high starch content (Caspar et al., 1991, Plant Physiol. 95: 1 181-1 188; Trethewey and ap Rees (1994) Biochem. J. 301, 449-454). This mutant is principally due to its enzyme content It has been shown that the TC26 mutant has a functional TPT, but cannot transport glucose as a product of starch degradation from the chloroplasts into the cytosol. The supposed lack of the glucose translocator leads to an accumulation of the Starch degradation products and thus a new synthesis of starch combined with the observed high starch content. Taken together, the experiments discussed underline the decision The glucose translocator is extremely important in the export of carbon frameworks from starch degradation. The glucose translocator was first described in 1977 (Schäfer et al., 1977, Plant Physiol 60: 286-289), but a more extensive biochemical or molecular characterization of this transport system is pending.

The process of photoassimilate production described and taking place in the leaves (the "source" tissue) serves to a large extent to supply non-green organs (the "sink" tissue), such as root, tuber and fruit, including the harvestable parts of the plants. In crops such as potatoes, sugar beets and cereals, the transport of carbohydrates from the "source" to the "sink" tissues via the sieve tubes (the phloem) takes place exclusively in the form of sucrose. The sucrose is then transported from the phloem into the cells of the "sink" tissue (phloem discharge). The transport can either be direct, that is to say symplastic, via connections (plasmodesms) existing between the phloem and the “sink” tissue, or else apoplastically, which means that the sucrose is discharged into the cell wall and imported again into the target cells. Alternatively, the sucrose in the apoplast can be cleaved into hexoses by a cell wall-specific invertase, which are then transported into the cells of the "sink" tissue. If it is transported in the form of sucrose, it is split into hexoses (glucose and fructose) in the cytoplasm of the cells of the "sink" tissue. This is done either via invertases or via sucrose synthase. All sucrose cleavage products are ultimately converted back into hexose phosphates (glucose-6-phosphate, glucose-1-phosphate).

In most plants, glucose-6-phosphate then serves as a substrate for starch synthesis, the entire reaction sequence of which is located in the interior (stroma) of the amyloplasts of the "sink" tissue. These organelles are surrounded by two lipid bilayer membranes, of which the outer membrane is non-specifically permeable to smaller molecules due to the presence of pore-forming proteins (porins). Such a porin of the outer amyloplast envelope membrane has recently been identified by us and the sequence of its amino acid components that make up the protein has been elucidated (Fischer et al., 1994, J. Biol. Chem., 269: 25754-25760). The inner envelope membrane, on the other hand, is the actual permeability limit between the cytoplasm and the organelles and, like a priori any lipid bilayer, is in principle impermeable to larger charged molecules such as e.g. Hexose phosphate. This basic impermeability of the inner amyloplast envelope membrane for larger hydrophilic molecules is bridged by the presence of specific transport systems

Glucose-6-phosphate is imported into the plastids via a transporter recently characterized by us, the glucose-6-phosphate / phosphate translocator (GPT) (Kammerer et al. (1998) Plant Cell 10: 105-1 17). Within the plastid, the imported glucose-6-phosphate can then be fed into the starch biosynthesis. The amyloplasts are transported in a counter-exchange mechanism, here in exchange with inorganic phosphate, which is released in the course of starch synthesis. Glucose-6-phosphate is converted into glucose-1-phosphate, which is then converted into ADP-glucose, the substrate for the starch synthase, via the ADP-glucose pyrophosphorylase (AGPase). Recent work has shown that in the endosperm of some cereals (barley, maize) the AGPase is also located in the cytosol and that the precursors for starch biosynthesis can be transported via ADP-glucose (Denyer et al., 1996, Plant Physiol. 12: 779-785). An exception to this may be fat-storing plastids from castor bean common. Here there is evidence that glucose is taken up from the cytosol directly into the plastids via a glucose transporter and there is phosphorylated to glucose-6-phosphate by a plastid hexokinase. (Dennis and Miernyk, 1982, Ann. Rev. Plant Physiol. 33: 27-50).

The imported glucose-6-phosphate is also the starting substrate for the oxidative pentose phosphate pathway (OPPP), the reduction equivalents, mainly for the reduction of the nitrite formed from the nitrate to ammonium, which in turn is removed into the biosynthesis of amino acids / proteins, provides. In this case, glucose-6-phosphate is transported in exchange with triose phosphates, the products of OPPP (Kammerer et al. (1998) Plant Cell 10: 105-1 17).

The starch formed in the non-green plastids, like the assimilation strength of the chloroplasts, is subject to constant build-up and breakdown (Stitt and Heldt, 1991, Biochim. Biophys. Acta 638: 1 -1 1). If the rate of synthesis is higher than that of degradation, the starch is created as a long-term storage for the fixed carbon. The starch 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 is remobilized and the starch degradation products then serve as fuel for the resulting seedling. It can be assumed that, as explained above using the example of the degradation of chloroplast assimilation starch, the glucose translocator of the non-green plastids plays a decisive role in the mobilization of the storage starch.

As can be seen from the explanations, transporters - like the chloroplastic triosephosphate-phosphate translocator, which catalyzes the transport of the fixed carbon out of the chloroplast, the sucrose translocator, via which the sieve tubes are loaded with the transport form of the photoassimilate sucrose, are glucose -6-phosphate-phosphate translocator of the amyloplasts, which provides the starting substrate for the starch synthesis, or the glucose translocator, via which the degradation products of the Starch mobilization leaves the plastids at a crucial point in the allocation of the photoassimilate from the "source" to the "sink" organs. Changes in the activity of a specific membrane transporter can have a major impact on plant metabolic performance. It has recently been shown that the effectiveness of photosynthetic carbon reduction (light reaction and Calvin cycle) can be influenced significantly by the activity of the chloroplastic triosephosphate-phosphate translocator (see above). The reduction of the sucrose transport activity via an "anti-sense" inhibition of the sucrose transporter responsible for the transport into the sieve tubes (Riesmeier et al., 1992, EMBO J. 1 1: 4705-4713) also leads to one drastic phenotype of the plants: they are much smaller, the leaves are damaged and the plants, in the case of potatoes, hardly produce any tubers, ie 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 the transport of degradation products of starch hydrolysis from photosynthetically active and non-green plastids. If it was possible to clone this translocator and to change the activity of the translocator in plants using the sequence obtained, this opened e.g. the possibility of increasing the starch content of the plants and thus their dry matter.

As Stark et al. (Science 258: 287-292) showed in 1992 that the flow towards starch synthesis in potato tubers could be increased via the overexpression of an ADP-glucose pyrophosphorylase (AGPase), an amyloplastidischen enzyme of the starch biosynthesis. An enzyme from E. coli was used for these experiments, which is not subject to the metabolic control over the 3-phosphoglycerate / phosphate quotient. However, more detailed analyzes of the transgenic plants showed that the increased carbon flow into the starch was associated with an increased starch. Turnover due to 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, ie the starch degradation and the further use and export of the degradation product glucose, must be prevented. Plants with a reduced activity of the plastid glucose translocator could no longer export the products of the amylolytic starch breakdown, glucose, from the plastids. Thus, the glucose would immediately be reintroduced into the starch structure or the further starch breakdown would be inhibited by its glucose product and the starch content would be increased. Such a procedure can also be derived from the investigations described above for the high-starch mutant TC265, which has a high starch content due to the presumed deficiency in the glucose translocator.

An increase in the starch content of the potato tuber would be important both for the starch processing industry and for the food industry, since potatoes with an increased starch content absorb less fat during their processing into chips, French fries, pastries etc. and therefore lead to lower-calorie end products. Successful strategies would most likely also be to use other starch-storing plants such as Corn, wheat and barley transferable.

If it were possible to produce potatoes with a reduced activity of the glucose translocator, storage losses caused by the so-called "cold sweetening" could also 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. Due to the Maillard reaction that occurs, the formation of reducing sugars is an extremely negative parameter during the further processing of the potatoes, such as the production of chips, etc. The formation of soluble sugars - hydrolysis products of the starch that accumulate in the extraplastid compartment - could be prevented by switching off the glucose translocator, which is involved in the removal of the starch-derived products, during the storage phase of the potatoes. This effect could be reinforced by a simultaneous reduction of the glucose-6-phosphate / phosphate Translocator that exports the phosphorylated C3 bodies from the phospholytic starch degradation from the plastids (Kammerer et al., 1998, Plant Cell 10: 105-1 17).

As explained above, the photoassimilates formed during photosynthesis are mainly exported during the day via the TPT. 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, in particular 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, in particular with a simultaneous overexpression of the ADP-glucose pyrophosphorylase, which leads to an increased starch turnover due to an increased breakdown of the starch (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 requires a corresponding presequence ("targeting sequence") which directs the attached mature protein to the plastids correctly (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 Sei. 96: 351-354). In addition to the presequence required for "plastid addressing", the mature part (the part of the protein which remains after the presequence has been cleaved off by a specific protease) of the plastid envelope membrane proteins contains further information which is necessary for the specific insertion of the proteins into the membrane are responsible and prevent transport of the envelope membrane proteins over the envelope membrane into the plastid stroma or, in the case of the 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). Our own work also showed, using the example of a mitochondrial carrier, the ADP / ATP transporter, that this protein cannot or only with very little efficiency can be directed to plastids and built into the envelope membrane there. Even a hybrid protein, consisting of a plastid presequence (containing the information for plastid addressing) and this mitochondrial carrier, showed a hardly increased incorporation into the plastid envelope membrane compared to the authentic protein (Silva-Filho et al., 1997, J. Biol. Chem. 272: 15264-15269; unpublished observations). Since the protein / lipid interaction is important for a correct insertion and the plastid envelope membrane differs fundamentally in its lipid composition from that of other cell organelles and also bacteria (Joyard et al., 1991, Eur. J. Biochem. 199: 489- 509), it is very unlikely that an insertion, albeit a small one, of a heterologous protein will then also be functional, ie that a transporter from other systems can be built into the plastid envelope membrane at all in a conformation and orientation corresponding to its function .

Thus, according to the current state of the art, it is not possible to functionally integrate, for example, mitochondrial or prokaryotic transporters into the inner plastid envelope membrane using known plastid "targeting" sequences. In the current state of the art, it is more promising to obtain the 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 believed that its primary structure is very different from that of the plasma membrane glucose transporters. These transporters belong to a large 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).

As stated above, physiological and biochemical studies of the Λrab / ^' o'ops / ^' s high-strength mutant TC265 have led to the assumption that this mutant has a defect in the chloroplastic glucose translocator. An attempt was made to clone this gene using a card-based approach. The gene responsible for the mutation could be located on chromosome 1 by crossing the TC265 mutant (Columbia ecotype) with an Arabidopsis wüdtyp (Landsberg erecta ecotype) (own, unpublished studies). However, no sequence coding for a putative glucose translocator could be identified in this genomic DNA region.

In order to determine the primary structure of the plastid glucose translocator, the extremely difficult route of biochemical characterization, purification and isolation of the transport protein had to be followed in membrane proteins. Starting from chloroplastid envelope membranes made of spinach, this protein - using chromatographic methods in connection with substrate protection experiments compared to labeling with a radioactively labeled inhibitor N-ethylmaleimide - succeeded as a component of the inner chloroplast envelope membrane with an apparent molecular mass of approx. 43,000 Identify Dalton.

The translocator's N-terminus was found to be chemically modified and thus not accessible to N-terminal protein sequencing by automated Edman degradation. It was therefore necessary to isolate the protein in sufficient quantity by preparative SDS-polyacrylamide gel electrophoresis and to cleave it enzymatically with a protease (Lys-C) in order to determine internal peptide sequences (see exemplary embodiment 1). The following peptide sequence could be obtained in this way: (1) KGRSLEEIELALSPAV. It was possible to use the 3'-RACE method (rapid amplification of cDNA ends) and using degenerate (modeled after the obtained peptide) synthetic oligonucleotides to obtain a specific PCR fragment. This was used as a probe for the subsequent screening of a cDNA library from spinach leaves and it was possible to isolate a cDNA clone which isolated the complete cDNA (1864 base pairs) for the precursor protein of the chloroplastic glucose translocator with a molecular mass of 57.6 kDa contained (see embodiment 2). The spinach cDNA was then used to isolate the corresponding cDNA clones from tissues of Arabidopsis thaliana, Zea mays (maize), Solanum tuberosum (potato) and Nicotiana tabacum (tobacco). The integration of the DNA from Spinacia oleracea into the genome of the TC265 mutant did not lead to a complementation of the mutant phenotype (own, unpublished observations), which in turn proves that the gene coding for the glucose translocator is not by card-based cloning of the sex gene can be isolated. We were also able to show that the Arabidopsis thaliana glucose translocator gene we identified is located on chromosome 5. Another homologous Arabidopsis translocator gene was located on chromosome 1 approx. 4 cM above the sex7 locus (own, unpublished observations).

A comparison of the cDNA sequences obtained with sequences from the databases showed that there were only slight similarities with the known glucose transporters of the plasma membrane (<30%) and the homologies to bacterial hexose transporters were in the range of approximately 45%. There was also an approximately 73% homology to a hypothetical sugar transporter from Prunus armeniaca (apricot). The derived molecular mass of this transporter is approximately 49.8 kDa. Here, however, the wrong start codon was used for the translation of the DNA and it was overlooked that this protein has an additional N-terminal extension which could serve as a plastid addressing signal. An assignment to a cellular membrane or an organ part could therefore not be made for the gene identified in apricot. On the other hand, we were able to show that the chloroplastic glucose translocator is synthesized as a precursor protein, as well as post-translationally and with the N-terminal signal sequence being split off into the chloroplasts imported and inserted into the envelope membranes (see embodiment 3).

The present invention provides DNA sequences which come from a plant genome and code for plastid glucose translocators, the information contained in the nucleotide sequence leading to the formation and expression in plant cells of a ribonucleic acid and via this ribonucleic acid a glucose Translocator activity can be introduced into the cells or an endogenous glucose translocator activity can be suppressed. The invention particularly relates to the DNA sequences from Zea mays (maize), Solanum tuberosum (potato) and Spinacia oleracea (spinach) with the nucleotide sequences shown in the appendix (sequence protocols).

The invention furthermore relates to DNA sequences which hybridize with the DNA sequences listed in the appendix or parts thereof or derivatives which are derived from these sequences by insertion, deletion or substitution and code for a plastid protein which has biological activity of a glucose translocator.

Furthermore, the present invention relates to the use of the DNA sequences according to the invention or parts thereof or derivatives which are derived from these sequences by insertion, deletion or substitution, for the transformation of pro- and eukaryotic cells. In order to ensure the expression of the glucose translocator in transformed cells, the DNA sequences according to the invention can be introduced into vectors and combined with control elements for expression in prokaryotic or eukaryotic cells (see

Embodiments 3 and 5). Such control elements are, on the one hand, transcription promoters and, on the other hand, transcription terminators. The vectors can be used to transform eukaryotic cells with the aim of expressing a translatable messenger ribonucleic acid (RNA) which allows the synthesis of a plastid glucose translocator in the transformed cells, or with the aim of expressing a non-translatable, inversely oriented ("anti sense "), messenger ribonucleic acid, which synthesizes the endogenous Prevents glucose translocators. For this purpose, shorter fragments of the DNA sequences according to the invention with a relatively high degree of homology (more than approx. 65% homology) could also be used. Repression can also be achieved by overexpressing the homologous DNAs (co-suppression). The expression of endogenous glucose translocators can also be inhibited by the expression of a ribozyme constructed for this purpose using the DNA sequences according to the invention; also via insertion mutagenesis - or, as soon as the corresponding technology is available, by homologous recombination ("knock-out" mutant). A tissue-specific controlled expression of an RNA in accordance with the sequences according to the invention of a plant glucose translocator enables a change in the plant carbon metabolism, the economic importance of which is that an improved derivation of the photoassimilates from the chloroplasts is achieved or the breakdown products of the starch mobilization in the plastids be held back.

Plants can thus be produced which have an increased starch content and / or - in the case e.g. the potato - show reduced storage losses. This modification increases the nutritional value of plants and thus their economic value. The respective effects could possibly be increased if the tissue-specific expression of the glucose translocator in the leaf or in heterotrophic "sink" tissue were combined with the other translocators / enzymes of carbon / nitrogen metabolism (simultaneous expression of several target proteins such as eg when increasing the starch content or reducing storage losses of the potato).

Furthermore, the heterologous expression of the sequences described in split yeasts (embodiment 4 and Loddenkötter et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2155-2159) enables structural-functional studies of the glucose translocator which ultimately lead to the development of a specific one Inhibitor of this protein could result; Herbicide development in particular should be considered here, since the inhibition of a protein with a key function in the metabolic process would necessarily be lethal for the plant. Further these structure-function studies are the basis for a directed mutagenesis of the substrate binding site of the translocator in order to specifically change the substrate specificity of the transporter.

Methods for the genetic modification of dicotyledonous and monocotyledonous plants are already 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, these must be linked with transcriptional regulatory elements. Such elements, called promoters, are known (inter alia, Koster-Toepfer et al., 1989, Mol. Gen. Genet. 219: 390-396). Furthermore, the coding regions must be provided with a transcription termination signal 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 native or homologous as well as foreign or heterologous to the host plant. Termination areas are interchangeable. The DNA sequence of the transcription start and termination regions can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components. In preparation for the introduction of foreign genes into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker which allows selection of the transformed cells. Examples of vectors are pBR322, pUC series, M13 mp series, pACYC 184 etc. Depending on the method of introducing desired genes into the plant, further DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant, at least the right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA must be added as a flank region to the genes to be introduced. The use of T-DNA for the transformation of plant cells has been intensively investigated and adequately described (Hoekema, in: The Binary Plant Vector System, Offset-drukkerij Kanters BV. Ablasserdam, 1985, Chapter V; Fraley et al., Critic. Rev. Plant Sc. 4: 1-46; An et al., 1985, EMBO J. 4: 277-287). Once the DNA has been integrated into the genome, it is generally stable there and is also retained in the offspring of the originally transformed cell. It usually contains a selection marker that gives the transformed plant cells resistance to biocides (such as Basta) or antibiotics (such as kanamycin, bleomycin or hygromycin). The individually introduced marker will therefore allow the selection of transformed cells from cells that lack the inserted DNA.

In addition to transformation using agrobacteria, many other techniques are available for introducing DNA into a plant host cell. These techniques include protoplast transformation, microinjection of DNA, electroporation, and ballistic methods. Whole plants can then be regenerated from the transformed plant material in a suitable selection medium. The plants obtained in this way can then be tested for the presence of the introduced DNA using conventional molecular biological methods. These plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties.

The DNA sequences according to the invention (or derivatives or parts of these sequences) can also be introduced into plasmids which permit mutagenesis or a sequence change by recombining DNA sequences in prokaryotic or eukaryotic systems. This can change the specificity of the glucose translocator e.g. towards an affinity for fructose or galactose.

The glucose translocator could also be made insensitive to specific herbicides. With the aid of standard methods (Sambrock et al., 1989, Molecular cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA), base exchanges and / or base deletions can be carried out and / or synthetic or natural sequences can be added . Adapters or links can be used to connect the DNA fragments to one another. Manipulations which provide suitable restriction sites or which remove unnecessary DNA can also be used. Wherever insertions, deletions or substitutions such as transitions or transversions come into question can be used in v / fro mutagenesis, "primer repair", restriction or ligation. Sequence analysis, restriction analysis and other biochemical-molecular biological methods such as, for example, the expression of the modified protein in yeasts and the measurement of the modified transport properties in artificial liposomes (see

Embodiment 4; Loddenkötter et al., 1993, Proc. Natl. Acad. Be. USA 90: 2155-2159; Fischer et al., 1997, Plant Cell, 9: 453-462; Kammerer et al., 1998, Plant Cell 10: 105-117) or measuring the modified transport properties of the protein expressed in transgenic plants using a method recently developed by us for this purpose (Flügge and Weber, 1994, Planta, 194: 181 -185) applied.

The DNA sequences according to the invention (or parts or derivatives of these sequences) can be used according to

Standard methods (in particular hybridization screening of cDNA libraries with low stringency using the DNA sequences according to the invention or parts of the DNA sequences as a probe or the creation of probes for stringent and low-stringent screening strategies by deriving degenerate and / or non-degenerate ones Isolate primers from the DNA sequences according to the invention for PCR experiments with DNA or cDNA from other plants) from the genome of plants, similar sequences which code for proteins which also transport glucose.

The DNA sequences according to the invention contain regions in the translated protein which are capable of specifically directing the proteins synthesized on ribosomes in the cytoplasm to plastids and of preventing the occurrence of the proteins in other membrane systems of the cell. The protein region which directs the protein encoded by the DNA sequences according to the invention to plastids is in each case within the first 90 amino acids of the protein, is not necessary for the transport function of the protein and is removed after the protein has been successfully inserted into the plastid envelope membrane. By replacing these "plastid targeting" sequences with one of the known "targeting" sequences, for example for mitochondria, the translocator protein could be transferred to another membrane system direct eukaryotic cells and could possibly change the transport properties over the respective membrane. Likewise, the "plastid targeting" sequences of the glucose translocator or endogenous regions of the mature protein could be used to direct foreign proteins (eg bacterial transport proteins or transporters from yeasts) into the plastids or into the plastid envelope membrane of plant cells.

For a better understanding of the exemplary embodiments on which this invention is based, the most important methods used are explained below.

1. Cloning procedure

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

The vector pEVP11 (Rüssel and Nurse, 1986, Cell 45: 145-153) was used for the transformation of yeasts.

For the plant transformation, the gene constructions were cloned into the binary vector pBinAR (Höfgen and Willmitzer, 1990, Plant Sei. 66: 221-230).

2. Bacterial and yeast strains The E. coli strain DH5α (Hanahan et al., 1983, J. Mol. Biol. 166: 557-580) was used for the pBluescriptSK (pBSC) phagemid and for pEVP1 1 and pBinAR constructs.

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: 871 1 -8720).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred into the agrobacteria by direct transformation using the method of Höfgen and Willmitzer (1988, Nucl. Acids Res. 16: 9877). The plasmid DNA of transformed agrobacteria was isolated by the method of Birnboim and Doly (1979, Nucl. Acids Res. 7: 1513-1523) and, after suitable restriction cleavage, analyzed for correctness and orientation by electrophoresis.

4. Plant transformation

For each transformation, 15 small leaves of a tobacco sterile culture were cut into pieces with an edge length of approx. 1 cm using a scalpel. The leaf pieces were transferred into MS medium with 2% glucose, into which the bacterial cells were then stirred into the medium with an inoculation loop from a transformed Agrobacterium tumefaciens-K \ tr bacterium grown under strict selection on agar plates. After 30 minutes of incubation, the leaves became incubated on MS medium with 1.6% glucose for two days in the dark 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. After a week's incubation at 25 ° C and 3,000 lux illuminance, the beta-lactyl concentration in the medium was reduced by half. Small shoots grew from the leaf pieces without a pronounced callus phase, which were transferred to an MS medium with 2% sucrose, 250 mg / l betabactyl and 100 mg / l kanamycin for root formation. After root formation, the saplings were transferred to earth culture.

Arabidopsis was transformed by the method of Bechtold et al., 1993, C.R. Acad. Be. 316: 1 194-1199.

Filing:

On June 10, 1998 and June 10, 1999, 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 pBSC-E43 / 30-3 (DSM 12243) Phageimid pBSC-Zm-pGT (DSM 12862)

Phageimid pBSC-St-pGT (DSM 12863)

Embodiment 1

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

The glucose translocator in the envelope membranes of spinach chloroplasts was identified by a combination of radioactive labeling experiments and substrate protection experiments. It could be shown that the transport of glucose into isolated chloroplasts could already be inhibited by very low concentrations of the sulfhydryl reagents pCMBS and NEM.

Isolated chloroplasts were mixed with the radioactively labeled sulfhydryl reagent N-ethylmaleimide (NEM) in the presence or absence of glucose, maltose and sorbitol. The chloroplast envelope membranes were then isolated, separated by SDS-PAGE and fluorographs were prepared from the fixed, stained and dried gels. It was shown that a protein with an apparent molecular mass of 43 kDa was differentially labeled in SDS-PAGE under the various conditions.

The protein identified in this way as a glucose translocator was then extracted from the chloroplast envelope membranes by extracting the membranes with a mixture of chloroform and methanol (2: 1) together with four other proteins. The highly lipid-containing extract was not directly accessible for separation in preparative SDS gels and had to be 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 cut out of the gel and cleaved in the gel matrix with the endoprotease Lys-C (Fresco, 1979, Anal. Biochem. 97: 382-386). The resulting peptides were made from eluted the gel and separated by means of HPLC (Eckerskorn and Lottspeicher, 1989, Chromatographia, 28: 92-94). The amino acid sequence of the purified peptide fractions was determined by automated Edman degradation in the gas phase (Eckerskorn et al., 1988, Eur. J. Biochem. 176: 509-519). Two degenerate oligonucleotide sequences encoding this amino acid sequence were derived from the amino acid sequence of one of the two peptides (peptide 1) and the oligonucleotide in question was prepared by in vitro DNA synthesis.

Embodiment 2

Cloning of the glucose translocator

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. PolyA ⁺ RNA was isolated from this and a cDNA library based on this 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). The first specific oligonucleotide in combination with an "anchor (dT) 15" primer was used as the primer for the first RACE reaction. The products obtained were subjected to a second PCR, the respective "nested" primers being used here. A specific PCR fragment (300 bp) could be obtained.

Approximately 300,000 clones of the cDNA library were probed with this PCR fragment (see embodiment 1). Clones reacting positively were purified by standard methods and after preparation of the amplified phage DNA from the purified plaques, the insert coding for the glucose translocator was obtained by EcoRI restriction digest and by Southern blot analysis, using the above-mentioned PCR fragment as a probe, verified. After in vivo excision of the phagemid from the phage with the aid of the filamentous helper phage ExAssist, the clones were analyzed by determining the DNA sequence (dideoxy method: Sanger et al., 1977, Proc. Natl. Acad. Be. USA 74: 5463-5467) and the primary structure of the glucose translocator was derived from this DNA sequence (clone pBSC-E43-30/3). The sequence of the peptide could be found in the overall sequence of the translocator. The spinach cDNA was then used to isolate the corresponding cDNA clones from Arabidopsis thaliana, Zea mays (maize), Solanum tuberosum (potato) and Nicotiana tabacum (tobacco) tissues by screening the corresponding low stringency cDNA libraries.

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 carried out using T7 RNA polymerase according to the manufacturer's instructions (Pharmacia). The subsequent in vitro translation was carried out in the reticulocyte lysate (Boehringer-Mannheim) and the postribosomal supernatant was used for protein transport in intact spinach chloroplasts. The experiment was carried out in the dark or in the light; the mixture 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). They were analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227: 680-685) and subsequent fluorography (Bonner and Laskey, 1974, Eur. J. Biochem. 46: 84-88). The pre-sequence of the glucose translocator was shown to correctly direct the attached mature protein to its target membrane, the inner chloroplast envelope membrane; it is split off during the import process by a specific protease and the mature protein is formed. In the dark the insertion of the translocator could be energized by the addition of ATP, while in the absence of ATP no import was observed. In the light, the energy for the import of the protein in the form of ATP could be provided via the photosynthetic phosphorylation become; under these conditions the import was independent of added ATP. However, if photosynthetic phosphorylation and the associated production of ATP were prevented by the addition of a decoupler such as CCCP, the protein import was also blocked. It was also shown that the mature protein was inserted into the inner envelope membrane in a proteae-resistant manner: addition of proteases (eg thermolysin) which cannot permeate the outer envelope membrane cannot attack the mature protein built into the inner membrane. Pretreatment of the chloroplasts with a protease (eg thermolysin) led to a complete loss of binding and the import of the translocator, which showed that the presequence ("targeting" sequence) of the translocator was initially specifically bound to receptors of the outer membrane in a first step must become. 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, the corresponding fragment was first produced by means of PCR with an oligonucleotide, which lies at the interface of presequence and mature protein. The fragment obtained in this way was inserted into the yeast expression vector pEVP11 and after amplification of the construct in E. coli in LiCI / PEG made competent (Ito et al., 1983, J. Bact. 153: 163-168) leucine synthesis deficient S pombe cells were 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, since the pEVP1 1- E43-30 / 3 construct gives yeast cells the ability to grow on leucine-free medium.

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

The insert was isolated by restriction digestion from the phagemid pBluescript-E43-30 / 3 (pBSC-E43-30 / 3), which contained the cDNA for the spinose glucose translocator as an insertion (see embodiment 2) and was converted into the vector pBinAR ( Höfgen and Willmitzer, 1990, Plant Sei. 66: 221-230). After amplification of the resulting construct pBinAR-E43-30 / 3 in E. coli, the construct was transformed into agrobacteria and these were then used to infect leaf segments of tobacco and potato.

The transformants obtained were examined with the aid of Southern blot analyzes for the presence of the intact, non-rearranged chimeric gene. The glucose transport activity was examined in comparison to control transformants (transformed with vector pBinAR without insertion), as were the C / N ratio, photosynthesis rate, starch content and growth.

Claims

claims

1. DNA sequences which contain the coding region of a plastid glucose translocator and which have the nucleotide sequences shown in the appendix, the sequence listing

Components of the claim are, as well as parts or derivatives of these DNA sequences which are derived from these DNA sequences by insertion, deletion or substitution and code for a plastid protein which has the biological activity of a glucose translocator, and further DNA sequences that with these

Hybridize DNA sequences or their parts or derivatives.

2. Plasmids and phagemids containing a DNA sequence or a part or derivative of the DNA sequences or a further DNA sequence according to claim 1.

3. Phagemid pBSC-E43 / 30-3 according to claim 2 deposited under the DSM number DSM 12243 as E. coli strain DH5α pBSC-E43 / 30-3 containing this phagemid.

4. Phagemid pBSC-Zm-pGT according to claim 2 deposited under the DSM number DSM 12862 as E. coli strain SOLR pBSC-Zm-pGT containing this phagemid.

5. Phagemid pBSC-St-pGT according to claim 2 deposited under DSM number DSM 12863 as E. coli strain DH5α pBSC-St-pGT containing this phagemid.

6. Bacteria containing a DNA sequence or a part or a derivative of the DNA sequences or a further DNA sequence according to claim 1 or a plasmid / phagemid according to one of claims 2 to 5.

7. yeasts containing a DNA sequence or a part or a derivative of the DNA sequences or a further DNA sequence according to claim 1 or a plasmid according to one of claims 2 to 5.

8. plant cells containing a plasmid according to claim 2.

9. Transformed plant cells and transgenic plants regenerated therefrom containing a DNA sequence or a part or a derivative of the DNA sequences or a further DNA sequence according to claim 1, wherein this DNA sequence or this part or the derivative thereof or the further DNA sequence was introduced into the plant cells as part of a recombinant DNA molecule.

10. Use of the DNA sequences or parts or derivatives of these DNA sequences or of the further DNA sequences according to claim 1 a) for introduction into pro- or eukaryotic cells, these sequences optionally with controls that control the transcription and translation in the cells ensure, are linked and lead to the expression of a translatable mRNA which brings about the synthesis of a glucose translocator, b) to the identification of insertion mutants, to homologous recombination or to the expression of a non-translatable RNA by means of an "anti-sense" effect , a cosupression or a ribozyme activity prevents the synthesis of one or more endogenous plastid glucose translocators in the cells, c) to change the carbon / nitrogen ratio in leaves or in heterotrophic tissues, in particular to increase the starch content, d) to lower the Sugar formation during the Starch mobilization, e) for the isolation of DNA sequences which code for a polypeptide which has the biological activity of a glucose translocator, f) which serve as "targeting" sequences in order to use them

Directing sequences of prokaryotic or eukaryotic proteins, in particular enzymes or proteins which catalyze the active or passive transport of metabolites across membranes, into the plastid envelope membrane, into the plastid stroma or into the thylakoids, g) wherein these sequences contain coding regions which are necessary for a mature one Encoding protein with the biological activity of a glucose 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 directed, h) for the identification of substances that have an inhibitory effect on the transport of hexoses across the inner plastid envelope membrane and / or i) for the isolation of corresponding genomic clones, in particular the use of the corresponding promoter regions or of promoter subregions for tissue-specific expression of genes .