DE19529696A1 - Transgenic plant cells and plants with increased glycolysis rate - Google Patents

Abstract

The invention concerns transgenic plant cells and plants having an increased glycolysis rate. The glycolysis rate is increased by the introduction and expression in plant cells of a DNA sequence which codes for an invertase, preferably a deregulated or unregulated invertase, and a DNA sequence which codes for a hexokinase, preferably a deregulated or unregulated hexokinase. The invention further concerns processes and recombinant DNA molecules for producing plant cells and plants having an increased glycolysis rate.

Description

The present invention relates to transgenic plant cells and plants with one compared to non-transformed Plants increased rate of glycolysis. The increase in gly kolyserate is achieved through the introduction and expression a DNA sequence for a cytosolic invertase preferably a deregulated or unregulated invertase co dated, as well as a DNA sequence for a cytosolic Hexokinase, preferably a deregulated or unregulated one Encoded hexokinase in plant cells. The invention also relates to methods and recombinant DNA molecules for the production of transgenic plant cells and plants, with increased glycolysis rate, as well as the use of DNA sequences, the proteins with the enzymatic activity an invertase or proteins with the enzymatic activi coding a hexokinase for the production of plants, which have an increased glycolysis rate.  

Due to the continuously increasing need for Le food that comes from the ever growing world population results is one of the tasks of biotechnological Research to increase the yield of utility plants to strive. One way to accomplish this consists in the targeted genetic engineering of the Me tabolism of plants. The goals are, for example Primary processes of photosynthesis that lead to CO₂ fixation ren, the transport processes involved in the distribution of the Pho toassimilate are involved within the plant as well Metabolic pathways leading to the synthesis of storage materials, e.g. B. of starch, proteins or oils.

For example, the expression of a prokaryotic asparagus has been described gin synthetase in plant cells, making it in the transgenic plants u. a. to an increase in biomass production is coming (EP 0 511 979). Also suggested was the expression of a prokaryotic polyphosphate nose in the cytosol of transgenic plants, which makes it in Kartof seedlings for an increase in yield in relation to the Bulb weight of up to 30% comes. Furthermore, in the EP-A2 0 442 592 the expression of an apoplastic invert tase in potato plants, which are also used for Er increasing the yield of such modified transgenic plants leads. Further attempts focused on the Modifi cation of the activities of enzymes involved in the synthesis of Sucrose as the main transport metabolite in the most plants are involved (see e.g. Sonnewald et al., Plant Cell and Environment 17, (1995), 649-658).

In WO 91/19896 it is further described that the increases Starch biosynthesis by overexpression of one of them gulated enzyme of ADP-glucose pyrophosphorylase into one increased allocation of sucrose to potato tubers leads, which are stored there in the form of strength.

While many of these applications deal with the steps employed either to form photoassimilates in  Leaves (see also EP 466 995) or with the Formation of polymers such as starch or fructans in storage organs of transgenic plants (e.g. WO 94/04692) exist until no promising approaches that describe what modifications in the primary metabolic pathways to increase the rate of glycolysis chen. An increase in the rate of glycolysis is e.g. B. for all those Processes in the plant matter for the larger men genes of ATP are required. This applies e.g. B. for many trans port processes across membranes by a membrane potential or a proton gradient are driven by the activity of an H⁺-ATPase is generated. Furthermore, one Increasing the rate of glycolysis is important for growth in Meristem, the switch from vegetative to generative Growth, as well as for the synthesis of various memories substances, in particular oils.

The invention is therefore based on the object of plant cells oils and plants with increased glycolysis rate and ver drive and DNA molecules available for their manufacture to deliver.

The solution to this problem is provided by the in the claims designated embodiments be provided.

Thus, the present invention relates to transgenic plants cells with one compared to non-transformed Plant cells increased the rate of glycolysis, which is due to reason for the introduction and expression of DNA sequences that code for a cytosolic invertase, as well as DNA-Se sequences coding for a cytosolic hexokinase an increase in invertase and hexokinase activity. The transgenic cells can each have one or more Contain DNA sequences for an invertase or a Encode hexokinase.  

It was surprisingly found that the introduction and Expression of DNA sequences necessary for a protein with the Encode the enzymatic activity of a hexokinase simultaneous introduction and expression of DNA sequences, that for a protein with the enzymatic activity of a Invertase encode one in the cytosol of plant cells drastic increase in the rate of glycolysis in such a changed Plant cells compared to unchanged plants cells can be reached.

Expression of an invertase fungal origin in the cytosol has already been described (see EP-A2 442 592). However, only the Expression of the fungal invertase in the plant cytosol Cells alone. The use of the was not described cytosolic invertase to increase the rate of glycolysis, ins especially in combination with the expression of a Hexoki nose.

An increased rate of glycolysis means that the transgenic Plant cells that have been transformed with DNA sequences those for the additional synthesis of an invertase and a Hexokinase result in the cytosol of the cells compared to non-transformed plant cells have an increased glycolysis have serate, preferably a glycolysis rate, um is increased by at least 30%, especially one that increases by at least at least 50% to 100%, and especially one that is around is increased more than 100% to 200%, preferably by more than 300%. Determining the rate of glycolysis by determination the corresponding metabolic intermediate is detailed described in the examples. The increase in glycol For example, serate can be determined by the concessions increase in glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, pyruvate or ATP. As part of the present ing invention, the determination of the increase in Glycolysis rate preferably by determining the increase Pyruvate.  

The provision of larger amounts of ATP through a gesti Lowered glycolysis rate, for example, enables the increase various energy-dependent transport and growth pro zesse. The provision of higher pyruvate concentrations than Result of an increased glycolysis rate leads to increased men gene on AcetylCoA, for example for the reinforced syn thesis of oils, fats or isoprenoid derivatives used can be. Are DNA sequences in the cells at the same time expresses the synthesis of polyhydroxyalkanoic acids possible, the AcetylCoA can also form suchi ger alkanoic acids are used.

In a preferred embodiment of the invention DNA sequences that are necessary for an invertase or encode a hexokinase to make DNA sequences necessary for a Coding invertase or hexokinase, which compared to nor sometimes invertases occurring in plant cells or hexokinases are deregulated or unregulated. Deregu Liert means that these enzymes are not in the same Be regulated like those in unmodified Plant cells normally formed invertase and hexo kinase enzymes. In particular, these enzymes are subject to others Regulatory mechanisms, d. H. they will not be in the same Extent due to the inhibito present in the plant cells ren inhibited or allosterically regulated by metabolites. Deregulated preferably means that the enzymes have a higher activity than endogenously expressed in plant cells Have invertases or hexokinases. Unregulated means in the context of this invention that the enzymes in vegetable Cells are not subject to regulation.

The DNA sequences for a protein with the enzymati encoding the activity of an invertase, it can both prokaryotic, especially bacterial, as also eukaryotic DNA sequences, i.e. H. DNA sequences from plants, algae, fungi or animal organisms deln. The term mushrooms are used in this context  also yeasts, especially those of the genus Saccharomy ces, such as B. Saccharomyces cerivisiae. In the by the Sequences encoded enzymes can be either known act in nature occurring enzymes that a different have appropriate regulation by various substances, especially through the plant invertase inhibitors, as well as enzymes by mutagenesis of DNA sequences, for known enzymes from bacteria, algae, fungi, animals or encode plants.

In a preferred embodiment of the present invention the DNA sequences that are required for proteins with the en encode zymatic activity of an invertase, from fungi. The DNA sequences originating from fungi or from them Coded enzymes have the advantage that they are ver same to vegetable invertases not by vegetable Invertase inhibitors are regulated. To be favoured DNA sequences from Saccharomyces cerevisiae used the code for the invertase.

These sequences are known and (see. Taussig et al., Nucleic Acids Res. 11, (1983), 1943-1954; EP-A2 0 442 592). To localize the Ensure invertase in the cytosol of the plant cells len, existing DNA sequences necessary for Coding signal peptides, deleted, and the coding If necessary, give the region a new start codon (see EP-A2 0 442 592). In addition to the ge called DNA sequence from Saccharomyces cerevisiae also white tere DNA sequences for proteins with the enzymatic Coding the activity of an invertase (see EMBL Accession number X67744 S. xylosus, M26511 V. alginolyticus, L08094 Zymomonas mobilis, L33403 Zymomonas mobilis, U16123 Zea mays, U17695 Zea mays, X17604 S. occidentalis, Z22645 S. tuberosum, Z21486 S. tuberosum, M81081 tomato, Z35162 V. faba, Z35163 V. faba, D10265 V. radiata, Z12025 L. esculentum, Z12028 L. pimpinellifolium, Z12026 L. pimpinellifolium, X73601 A. sativa, 570040 acid invertase, V01311 yeast gene, U11033 Arabidopsis thaliana, X81795 B.  vulgaris BIN35, X81796 B. vulgaris, X81797 B. vulgaris, X81792 C. rubrum, X81793 C. rubrum, X77264 L. esculentum, Z12027 L. esculentum, D10465 Z. mobilis, D17524 Zymomonas mobilis) and also due to their properties Production of the plant cells according to the invention used can be, taking care that the Protein is formed in the cytosol of the plant cell. Techniques for modifying such DNA sequences to match the Localization of the synthesized enzymes in the cytosol of the Ensure plant cells are the expert knows. In the event that the invertases contain sequences, for secretion or for a specific subcellular loca lization are necessary, e.g. B. for localization in the extrazel lular space or the vacuole, the corresponding DNA sequences can be deleted.

The DNA sequences for a protein with the enzymati encoding the activity of a hexokinase, it can both prokaryotic, especially bacterial, as also eukaryotic DNA sequences, i.e. H. DNA sequences Act plants, algae, fungi and animal organisms.

Hexokinases (EC 2.7.1.1) are enzymes, the following reaction catalyze:

Hexose + ATP ↔ Hexose Phosphate + ADP

The hexokinases encoded by the DNA sequences can they are both known enzymes found in nature act a different regulation by different Have substances as well as enzymes caused by mutage nese from DNA sequences for known enzymes from bacteria encode algae, fungi, animals or plants were put. DNA sequences necessary for enzymes with Hexokina coding activity are from a number of organizations nisms described, for example from Saccharomyces cerevisiae, humans, rats and various microorganisms (for the DNA sequences see: EMBL gene bank accession numbers M92054, L04480, M65140, X61680, M14410, X66957, M75126, J05277, J03228, M68971, M86235, X63658).  

In a preferred embodiment it is DNA sequences encoding glucokinases, especially glucoki noses with reduced allosteric regulation, through z. B. glucose-6-phosphate, are subject. Glucokinases (E. C 2.7.1.2) are hexokinases with a high affinity for Glucose that catalyzes the following reaction:

Glucose + ATP ↔ Glucose-6-Phosphate + ADP

In a preferred embodiment, the DNA sequences originate zen which code for a glucokinase from Zymomonas mobilis (Barnell et al., 1990, J. Bacteriol. 172, 7227-7240; EMBL- Genbank accession number M60615). Other glucokinases are from humans and rats (for the DNA sequences see: EMBL gene bank accession numbers M69051, M90299, J04218 and M25807).

Furthermore, DNA sequences that are necessary for an invertase or encode a hexokinase with the help of the be knew the above-mentioned DNA sequences from any organization nisms are isolated. Methods for isolation and Identification of such DNA sequences is known to the person skilled in the art common, for example hybridization with known Sequences or by polymerase chain reaction using extension of primers derived from known sequences are.

The enzymes encoded by the identified DNA sequences are then in terms of their enzyme activity and Regulation examined.

By introducing mutations and modifications after Techniques known to those skilled in the art can be carried out by DNA Se sequences further encoded proteins in their regulatory Properties can be changed to de- or unregulated To get enzymes.

For expression in plant cells, the DNA Se sequences for a cytosolic invertase or hexokinase  encode, in principle under the control of any in plant cells are functional promoter. The ex pression of said DNA sequences can generally be in any Tissue from a transformed according to the invention Plant cell regenerated plant and at all times take place, but preferably takes place in such tissues instead, in which an increased glycolysis rate is advantageous neither for the growth of the plant, for the uptake and the Transport of ions and metabolites or for the bil of ingredients within the plant. Suitable Therefore, mainly promoters appear that have a specifi cal expression in a certain tissue, to a be agreed with the time of development of the plant or in a certain organ of the plant. Especially suitable for increasing fatty acid biosynthesis as a result an increased AcetylCoA content in seeds of oil-producing Plants like rapeseed, soybean, sunflower and oil palms therefore appear to be promoters that are specific in the endosperm or but are active in the cotyledons of seeds that form. Such promoters are e.g. B. the phaseolin promoter Phaseolus vulgaris, the USP promoter from Vicia faba or the HMG promoter from wheat.

In a further preferred embodiment, the DNA sequences under the control of promoters that have a Ensure seed-specific expression. In case of Starch-storing plants, such as B. of corn, wheat, Barley or other cereals will be in the seeds Glycolysis rate increases and there is an increased formation of Pyruvate and AcetylCoA and an enhanced fatty acid biosyn these instead. This means a change in the flow the photoassimilates from starch towards pyruvate dependent biosynthetic pathways, such as. B. the fatty acid biosyn thesis.

In a further preferred embodiment, Ex pression of the DNA sequences used in promoters  Storage organs such as tubers or roots are active, e.g. B. in the root of the sugar beet or in the tuber the potato. In this case, it comes to expression of the DNA sequences for an invertase or hexokinase encode, to redirect biosynthetic pathways in the sense the formation of less sugar or starch and an increase formation of pyruvate and acetyl-CoA due to the increased Glycolysis rate.

Furthermore, the expression of the DNA sequences under the Kon trolls are made by promoters that are specific at the time the flowering induction are activated or are active in Tissues that are necessary for flower induction. As well can be used by only one promoter external influences controlled time are activated, e.g. B. by light, temperature, chemical substances (see for example WO 93/07279). For increasing the uptake rate of ions from the soil due to an increased ATP content are z. B. promoters of interest, a root hair or Root-epidermis-specific expression. For the increase in the export rate of photoassimilates from the Leaf are e.g. B. Promoters of interest who have an escort have cell-specific expression. Such promoters are known (e.g. the promoter of the rolC gene from Agrobacte rium rhizogenes).

In a preferred embodiment, the DNA sequences are which code for an invertase or hexokinase, except with a promoter, linked to DNA sequences that a white Ensure a further increase in transcription, for example wise so-called enhancer elements or with DNA sequences, which are in the transcribed area and which are efficient tere translation of the synthesized RNA into the corresponding ensure proper protein. Such regions can range from viral genes or suitable plant genes or manufactured synthetically. They can be homologous or be heterologous to the promoter used. Advantageously  are the DNA sequences necessary for an invertase or a Encode hexokinase with 3'-untranslated DNA Se linked sequences that terminate the transcription and ensure the polyadenylation of the transcript. Derar term sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens. These sequences are interchangeable.

The DNA sequences for an invertase or hexokinase encode, are in the plant cells according to the invention preferably stably integrated into the genome.

In the transgenic plant cells, which due to the additional expression of a cytosolic invertase and a cytosolic hexokinase showed an increased rate of glycolysis point, it can basically be any cell act against plant species. Both Zel len monocotylers as well as dicotyled plant species, esp special cells that store starch, oil or land economic crops, such as B. rye, oats, ger ste, wheat, potato, corn, rice, rapeseed, pea, sugar beet, Soybean, Tobacco, Cotton, Sunflower, Oil Palm, Wine, To mate etc. or cells of ornamental plants.

The plant cells according to the invention, the increased Glycolysis rate can for the regeneration of whole, in clock plants are used. Subject of the present the invention are thus also transgenic plants, which he are preserved by regeneration of an inventive Plant cell, as well as plants, the trans contain gene plant cells.

By providing plant cells with a ge increased rate of glycolysis it is now possible to transgene Plants with changed advantageous properties put. For example, by increasing the Glycolysis rate specific in escort cells of transgenic plants  loading the sieve element-guide cell complex with Sucrose through the sucrose-proton cotransporter be increased, resulting in an increase in the transport rate of Photoassimilates leads. In the same way, the recording of inorganic ions such as phosphate, sulfate and. a. from the Soil above the roots.

An increase in the rate of glycolysis specifically in root cells len, especially in root hairs and epidermal cells due to the increased H⁺-ATPase activity to a ver lead to increased excretion of protons in the soil. A such acidification of the soil leads to mobilization and thus facilitated absorption of various minerals, such as e.g. B. Phosphate from the soil.

Of particular importance is the possibility of increasing the rate of glycolysis in the oil-storing tissues of a plant, such as B. the endosperm or the cotyledons of seeds or in other oil-storing organs, the flow of the seeds or organs delivered Photoassimilate in the direction of To direct formation of pyruvate and AcetylCoA. The ready development of increased amounts of these precursors for the Biosyn thesis of triglycerides leads to an increased synthesis of oils and thus to an increased yield. A ready Providing increased amounts of AcetylCoA is also common for many other processes naturally occurring in plants such as the isoprenoid biosynthesis, but on for the formation of Po important such as polyhydroxyalkanoic acids (see e.g. Poivier et al., Bio / Technology 13, 1995, 142-150).

By increasing the rate of glycolysis in starch-storing Tissues, for example in seeds of various cereals or in the tubers of the potato, there is a Change in the flow of photoassimilates from starch biosynthesis towards pyruvate-dependent biosynthetic pathways, for example fatty acid biosynthesis. The result is a decrease in the amount of starch in the corresponding Ge  weave with possible simultaneous increase in fatty acid biosynthesis.

It is also important that the increase of the glycolysis rate produced high levels of AcetylCoA also to increased isoprenoid synthesis or, in Kombina tion with the expression of appropriate genes for synthesis from polyhydroxyalkanoic acids to a polyhydroxyalkanoic acid Synthesis in the transgenic plant cells can lead.

The present invention further relates to a method for Production of transgenic plant cells compared to non-transformed plant cells have an increased glyco have lysis rate, in which in plant cells DNA Se sequences are introduced which are necessary for a cytosolic invert encode tase, as well as DNA sequences necessary for a cytosoli coded hexokinase, and these sequences in the trans formed plant cells are expressed.

Such a method preferably consists of the fol steps:

• (a) Preparation of a recombinant double-stranded DNA molecule which comprises the following DNA sequences:
  • (i) a promoter that ensures transcription in plant cells;
  • (ii) a DNA sequence which codes for a protein with the enzymatic activity of an invertase and is linked in the sense direction to the promoter;
• (b) the production of a recombinant double-stranded DNA molecule which comprises the following DNA sequences:
  • (i) a promoter that ensures transcription in plant cells;
  • (ii) a DNA sequence which codes for a protein with the enzymatic activity of a hexokinase and is linked in the sense direction to the promoter; and
• (c) Transfer of those prepared according to step (a) and (b) DNA molecules in plant cells.

For the choice of plant species, promoters, others flanking DNA sequences, as well as for choice and modification cations of the DNA sequences for an invertase or a Coding hexokinase is the same as in the previous section in connection with the cells according to the invention has been.

The DNA sequences for an invertase or a hexoki nose encoding can either be on separate DNA molecules be localized, or together on a recombinant DNA molecule. The sequences are on two different those DNA molecules, the transfer of the DNA molecules can ent not done simultaneously, or in such a way that plant cells are first transformed with a DNA molecule and se then read plant cells with the second DNA molecule to be transformed. Furthermore, plants that both an additional cytosolic invertase and one express additional cytosolic hexokinase by first creating two independent transgenic plants lines are created that an invertase or a hexo encode kinase and then cross them.

The transfer of DNA molecules that contain DNA sequences which code for invertase or hexokinase, occurs before preferably using plasmids, especially sol Chen plasmids that have a stable integration of the DNA mole Ensure plant cells transformed into the genome most, for example binary plasmids or Ti plasmids of Agrobacterium tumefaciens system. In addition to the Agrobacterium System come other systems to introduce DNA molecule len in plant cells in question, such as. B. the so-called biolistic processes or the transformation of Pro toplasts (cf.Willmitzer L. (1993), Transgenic Plants, Biotechnology 2; 627-659 for an overview). To the Transfor Basically, cells of all plant species come in Question. Both monocot and diko are of interest  tyle plants. For various monocot and dicot styles Plant species are already transformation techniques been written. Cells are preferred in the processes agricultural crops used, in particular of cereals, e.g. B. rye, oats, barley, wheat, kar potato, corn, rice, rapeseed, pea, sugar beet, soybean, ta bak, cotton, sunflower, oil palm, wine, tomato etc. or Ornamental plant cells. The subject of the invention are if the transgenic plant obtainable from the process cells and from them obtainable by regeneration Plants that due to the additional expression of a cy Tuscan invertase and a cytosolic hexokinase have an increase in the rate of glycolysis.

The present invention further relates to recombinant DNA Molecules that contain a DNA sequence required for a pro with the enzymatic activity of a hexokinase preferably a glucokinase, encoded, in combination with DNA Sequences that transcription and translation in plant ensure cells. The hexokinase is preferably a deregulated or unregulated one Enzyme.

The present invention further relates to recombinant DNA molecules that include the following DNA sequences:

• (i) a DNA sequence necessary for a cytosolic invertase encoded, in combination with DNA sequences that the Transcription and translation in plant cells guarantee; and
• (ii) a DNA sequence necessary for a cytosolic hexokinase encoded, in combination with DNA sequences that the Transcription and translation in plant cells guarantee.

For the choice of the DNA sequences, the transcription and Allow translation in plant cells applies to  recombinant DNA molecules the same as that already mentioned above Connection with the cells and methods according to the invention was carried out, as well as for the choice of DNA sequences, which code for an invertase or hexokinase.

Finally, the present invention relates to uses DNA sequences coding for an invertase, preferably for a deregulated or unregulated, for Production of transgenic plant cells compared to non-transformed plant cells have an increased glyco exhibit lysis rate. The invention also relates to Use of DNA sequences which are suitable for a hexokinase co dieren, preferably for a deregulated or unregu giert, for the production of transgenic plant cells, the compared to non-transformed plant cells have increased glycolysis rate.

Description of the picture

Figure 1 shows the 12.68 kb plasmid pB33Hyg-GK. The plasmid contains the following fragments:

A = fragment A (1498 bp) contains the DraI-DraI fragment (Position -1512 to position +14) of the promoter region of the Patatin Gens B33 (Rocha-Sosa et al. (1989) EMBO J. 8, 23-29).

B = fragment B (1025 bp) contains a DNA fragment with the Coding region of Zymomonas mobilis glucokinase (Genembl accession number: M60615; nucleotides 5128 to 6153).

C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, Nucleotides 11749-11939.  

Methods 1. Cloning procedure

The vector pUC18 was used for cloning in E. coli det. For the plant transformation, the gene cones structures in the binary vector pBinAR (Höfgen and Willmitzer (1990) Plant Sci. 66, 221-233).

2. Bacterial strains

For the pUC vectors and for the pBinAR constructs the E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh, USA) used.

The transformation of the plasmids into the potato plants was made using the Agrobacterium tumefaciens strain C58C1 pGV2260 performed (Deblaere et al. (1985) Nucl. Acids Res. 13, 4777-4788).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transforma tion according to the method of Höfgen and Willmitzer ((1988), Nucleic Acids Res. 16, 9877). The plasmid DNA transfor mated agrobacteria was made using the method of Birnboim and Doly ((1979), Nucleic Acids Res. 7, 1513-1523) isolated and after a suitable restriction cleavage analyzed by gel electrophoresis.

4. Transformation of potatoes

Ten small leaves wound with a scalpel one Sterile potato culture (Solanum tuberosum L. cv. Desir´e) were in 10 ml MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473) with 2% sucrose which 50 ml of a grown under selection  Agrobacterium tumefaciens overnight culture contained. After Another shaking was carried out for 3-5 minutes Incubation for 2 days in the dark. Then the Callus induction leaves on MS medium with 1.6% Glu cose, 5 mg / l naphthylacetic acid, 0.2 mg / l benzylaminopu rin, 250 mg / l claforan, 50 mg / l kanamycin and 0.80% Bacto agar laid. After a week's incubation at 25 ° C and 3000 lux the leaves were on for sprout induction MS medium with 1.6% glucose, 1.4 mg / l zeatin ribose, 20 mg / l naphthylacetic acid, 20 mg / l giberellic acid, 250 mg / l Claforan, 50 mg / l Kanamycin and 0.80% Bacto Agar placed.

5. Radioactive labeling of DNA fragments

The radioactive labeling of DNA fragments was carried out with Using a DNA random primer labeling kit from the company Boehringer (Germany) according to the manufacturer carried out.

6. Plant husbandry

Potato plants were grown in the greenhouse under the following Conditions kept:

Light period: 16 h at 25000 lux and 22 ° C
Dark period: 8 h at 15 ° C
Air humidity: 60%.

7. Determination of starch content and dry matter of potato tubers

The determination of the starch content and the dry sub The potato tuber was punched using the Bestim specific gravity (Scheele et al. (1937) Landw. Vers. Sta., 127, 67-96) according to the following For meln:

% Dry matter = 24.182 + 211.04 × (specific weight - 1.0988)
% Strength = 17.546 + 199.07 × (special weight - 1.0988)

8. Determination of phosphorylated metabolic intermediates in Potato tubers

The determination of phosphorylated metabolic intermediates in potato tubers was made with minor changes after Weiner and Stitt ((1987) Biochim. Biophys. Acta, 893, 13-21).

Presentation of the potato tuber extract

Approx. 200 mg of tuber material were in each case underflown Homogenized nitrogen in a mortar. The phosphorylated intermediates were with 16% tri chloroacetic acid solution extracted in diethyl ether. After three hour incubation on ice was by three times Extract the trichloroacetic acid with diethyl ether distant. The extracts were then treated with 5 M KOH / 1 M Triethanolamine neutralized. The determination of Phosphoenolpyruvate (PEP) and pyruvate took place immediately. To determine the other intermediates, the Ex can be stored for several days at -70 ° C.

Determination of the phosphorylated intermediates followed on a two-wavelength photometer (Sigma ZWS 11) by means of coupled enzymatic reactions Stitt et al. (Methods in Enzymology, 174, 518-552)

a) Determination of PEP and pyruvate

The reaction buffer contained:

50 mM Hepes-KOH pH 7.0;
5 mM MgCl₂;
0.025 mM NADH;
1 mM ATP
Pyruvate determination: 1 U / ml lactate dehydrogenase
PEP determination: +2 U / ml pyruvate kinase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract.

b) Determination of UPD glucose (UDPG)

The reaction buffer contained:

200 mM glycine pH 8.7;
5 mM MgCl₂;
1mM NAD;
0.025 U / ml UDP-glucose dehydrogenase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract.

c) Determination of glucose-6-phosphate (G6P), fructose-6- Phosphate (F6P) and Glucose-1-Phosphate (G1P)

The reaction buffer contained:

50 mM Hepes-KOH pH 7.0;
5 mM MgCl₂;
0.25 mM NADP;
G6P determination: 2 U / ml glucose-6-phosphate dehydrogenase
F1P determination: + 2 U / ml phosphoglucoisomerase
G1P determination: + 2 U / ml phosphoglucomutase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract.

d) Determination of ATP

The reaction buffer contained:

50 mM Hepes-KOH pH 7.0;
5 mM MgCl₂;
0.25 mM NADP;
1 mM glucose;
2 U / ml glucose-6-phosphate dehydrogenase;
2 U / ml phosphoglucoisomerase;
1 U / ml hexokinase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract.  

e) Determined by ADP

The reaction buffer contained:

50 mM Hepes-KOH pH 7.0;
5 mM MgCl₂;
0.05 mM NADH;
0.2 mM PEP;
0.2 U / ml lactate dehydrogenase;
1 U / ml pyruvate kinase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract

f) Determination of 3-phosphoglycerate (3-PGA)

The reaction buffer contained:

100 mM Tris-HCl pH 8.1;
5 mM MgCl₂;
0.05 mM NADH;
1.33 mM ATP;
0.1 U / ml phosphoglycerate kinase;
2 U / ml glyceraldehyde phosphate dehydrogenase

The measurement is carried out at 25 ° C with 50 to 100 µl Ex tract.

9. Determination of the activity of carbohydrate enzymes metabolism in potato tubers

For the determination of enzyme activities (e.g. the glucoki nose, fructokinase, sucrose synthase, invertase, Phosphoglucomutase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, Phosphoglycerate mutase or pyruvate kinase) in potato 200 mg of tuber material were extracted in 500 µl extract tion buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl₂; 1 mM EDTA; 1mM EGTA; 1mM DTT; 2mM benzamidine; 2 mM ε- Amino-n-caproic acid; 0.5 mM PMSF; 10% (v / v) gly cerin; 0.1% (v / v) Triton X-100) homogenized. After centrifugation, 10 to 40 µl of the cell-free desalted extract for the enzyme activity measurement set. The enzyme activities were with the exception of Sucrose synthase and invertase activity by means of kop  pelter enzymatic reactions on a spectral photo meter determined.

The glucokinase and fructokinase activity was decreased Renz et al. ((1993) Planta, 190, 156-165), the sucrose- Synthase and invertase activity according to Zrenner et al. ((1995) Plant J., 7; 97-107), the phosphofructokinase, Glyceraldehyde-3-phosphate dehydrogenase, phosphoglyce ratkinase, phosphoglycerate mutase, pyruvate kinase stocks according to Burell et al. ((1994) Planta, 194, 95-101), and Pressey's phosphoglucomutase activity ((1967) Journal of Food Science, 32, 381-385).

The examples illustrate the invention.

example 1 Construction of the binary plasmid p33-Cy-Inv

The preparation of the plasmid p33-Cy-Inv and introduction of the Plasmids in the potato genome is in EP-A2 0 442 592 have been described. Regenerated potato plants with the binary construct p33-Cy-Inv has been transformed, were transferred to earth and regarding the invertase acti selected. Several genotypes were identified adorns the up to a hundred times higher invertase activi compared to the control plants, exhibit.

Example 2 Construction of the binary plasmid pB33Hyg-GK

The coding region of the Zymononas mobilis glucokinase gene using the polyme Lawn chain reaction (PCR) based on genomic Zymomonas mobilis DNA amplified. The sequence of the glucokinase  Zymomonas mobilis is in the GenEmbl database with the Accession number M60615 entered. The amplified question ment corresponds to the region from nucleotides 5128 to 6153 this sequence. An Asp718- Interface and at the 3'-end a HindIII interface added. The 1025 bp PCR fragment was over the two additional interfaces cloned into the vector pUCBM20. Starting from this plasmid, the entire coding region was the glucokinase after restriction digestion with EcoRI and HindIII subcloned into the vector pBluescriptSK. In extrac of E. coli cells that contain the resulting plasmid pSK-GK contained, was compared with extracts from untrans E. coli cells formed glucokinase acts increased 100-fold vity proven. For this, the cells were a 20 ml Harvested overnight culture and re right in 500 µl extraction buffer suspended (30 mM KH₂PO₄; 2 mM MgCl₂; 10 mM 2-mercaptoetha nol; 0.1% (v / v) Nonidet P40). After adding the same volume of acid-washed glass beads (0.1 mm diam ser) the suspension became violent four times for 30 seconds mixed up. After centrifugation, the glucokinase was activated vity in the cell-free extract as in Scopes et al. ((1985) Biochem. J., 228, 627-634).

After the functionality of the PCR product had been demonstrated in this way, the insert was recloned into a binary vector derived from pBIN19 (Bevan (1984) Nucl. Acids Res. 12, 8711-8720). The following plasmid was created: the plasmid pB33Hyg-GK (cf. FIG. 1). As a promoter for the expression of a transgene in plants, the construct contains the B33 promoter from Solanum tuberosum (Rocha-Sosa et al. (1989) EMBO J., 8, 23-29).

The construct pB33Hyg-GK was created as follows:
Since the construct should be used for the transformation of already transgenic potato plants which express the NPT-II gene, the plasmid pBIB, which contains the HPT gene coding for the hygromycin B phosphotransferase, was used (Becker (1990) Nucl. Acids Res. 18, 203).

The promoter of the B33 gene from Solanum tuberosum was named DraI fragment (position -1512 to +14 according to Rocha-Sosa et al. (1989) EMBO J. 8, 23-29) after removing protruding ends with Polymerase II in the SacI site of the plasmid pUC19 cloned. The promoter region became the EcoRI / SmaI fragment cloned into the binary vector pBIN19, which terminates signal of the octopine synthase gene from Agrobacterium tumefaciens in the immediate vicinity of a polylinker from M13mp19 contains. This resulted in pB33. The promoter bottom Lylinker terminator fragment of plasmid pB33 was identified as EcoRI / HindIII fragment in the linear with EcoRI and HindIII plasmid pBIB cloned. This gave rise to the plasmid pB33Hyg.

The coding region of the glucokinase was subsequently made Asp718 / SalI digestion of plasmid pSK-GK isolated and Asp718 / SalI cloned into plasmid pB33Hyg. From this resul plasmid pB33Hyg-GK, which for the transforma tion of the transgenic potato line U-Inv-2 (line 30) was set.

For the transformation of Agrobacterium tumefaciens the binary plasmid by direct transformation according to the method by Höfgen & Willmitzer ((1988) Nucl. Acids Res. 16, 9877) introduced into the cells. The plasmid DNA transformed Agrobacteria were determined using the method of Birnboim et al. ((1979) Nucl. Acids Res., 7, 1513-1523) isolated and after suitable restriction cleavage gel electrophoretic analy siert. For the transformation of potato plants at for example 10 small leaves wound with a scalpel a sterile culture in 10 ml MS medium with 2% (w / v) Sucrose placed, which 30 to 50 ul a Selek tion grown overnight Agrobacterium tumefaciens culture contains. After 3 to 5 minutes of gentle shaking the Petri dishes are incubated at 25 ° C in the dark. After 2 days  the leaves on MS medium with 1.6% (w / v) Glu cose, 2 mg / l zeatin ribose, 0.02 mg / l naphthylacetic acid, 0.02 mg / l giberellic acid, 500 mg / l claforan, 3 mg / l hygro mycin and 0.8% Bacto agar. After a week of inku bation at 25 ° C and 3000 lux becomes the Claforan concentration reduced by half in the medium. The further cultivation was carried out as described by Rocha-Sosa et al. ((1989) EMBO J., 8, 23-29).

Example 3 Analysis of transgenic potato plants that have an invertase from yeast and a glucokinase from Zymomonas mobilis in Express tubers

Regenerated potato plants of the U-Inv-2 (30) line, the were transformed with the binary construct pB33Hyg-GK have been transferred into earth and regarding the glucoki selected nose activity in tubers. There were several Genotypes identified that increased up to five times Glucokinase activity compared to the control plants, have (e.g. GK-41, GK-29, GK-38). Furthermore was demonstrated that these genotypes continue to have the invertase gene express from yeast (see Table I).

Table I

The enzyme activities shown here are the mean based on at least five measurements based on five independent against plants.

The above mentioned genotypes GK-41, GK-29 and GK-38 were amplified, and 15 plants each were grown house transferred. The tubers were harvested for 4 months later.

Surprisingly, it was found that the tubers of the transgenic plants GK-41, GK-29 and GK-38 only 40 to Contain 60% of the starch of control plants, whereby but the starch-free proportion of dry matter is constant remains (see Table II). This shows that only the Starch content in the tubers of the GK plants by the Ex pression of the invertase in combination with the expression of the Glucokinase is reduced. It also became an Er wearing depression was found to be 25%. This correlates like in turn with the reduction of the starch content.

Table II

Analysis of soluble sugars such as glucose, fructose and Sucrose surprisingly showed that seven times the An the glucose concentration in the U-Inv-2 plants increased compared with the wild type through the expression of glucokinase is greatly reduced so that the amount of glucose is only 30% the amount of glucose in tubers of WT control plants is. The fructose concentration is in the transgenic lines  not changed compared to the control plants. The strong re reduction in the amount of sucrose in the U-Inv-2 plants partially canceled out by the expression of glucokinase (see Table III). In summary, it was found that e.g. B. the tubers of the GK-38 plants only 40% starch and 50% soluble sugar compared to tubers from untransfor contained control plants.

Table III

Since there is no evidence that the amount of Photoassimi laten that over the phloem from the mature leaves in the Bulbs transported are reduced in the GK plants is, but on the other hand reduced contents of strength and soluble sugars are measured, it can be assumed that the expression of an invertase in the cytosol of plant cells in combination with the expression of a glucokinase in Cyto of plant cells to increase glycolysis and of respiration.

This surprising result is due to changed levels of metabolites and altered enzyme activities in tuberex tracts of the GK plants underlined. It became a five che increase in the glucose-6-phosphate content, a five-fold Increase in fructose-6-phosphate content, a 40% increase reduction of the 3-phosphoglycerate content, a sixfold increase increase in pyruvate content and a 50% increase in ATP content detected (see Table IV).  

Table IV

The metabolite amounts shown here are the average of at least five measurements based on five independent plants. The values are given in nmol g ^-1 fresh weight.

It has also been shown that the activity of enzymes, which reactions of glycolysis catalyze in Knollenex tracts of GK plants is increased (see Table V).

There are e.g. B. the specific activities of fructokinase, the phosphofructokinase, the glyceraldehyde-3-phosphate dehy drugase (GAP-DH) and pyruvate kinase increased.

Expression of an invertase in the cytosol of plant cells in combination with the expression a glucokinase in the cyto So of plant cells is a process which to increase glycolysis and respiration leads.  

Table V

The enzyme activities shown here are the average of at least five measurements based on five independent plants. The values are given in nmol min ^-1 mg ^-1 fresh weight.

Claims (34)

1. Transgenic plant cell with one compared to not transformed plant cells increased glycoly serate, due to the introduction and express sion of DNA sequences containing a cytosolic invertase encode, as well as DNA sequences that a cytosoli encoding hexokinase, to increase the Inver tase and hexokinase activity comes. 2. Transgenic plant cell according to claim 1, wherein the In vertase is a deregulated enzyme. 3. Transgenic plant cell according to claim 1, wherein the In vertase is an unregulated enzyme. 4. Transgenic plant cell according to one of claims 1 to 3, wherein the hexokinase is a deregulated enzyme. 5. Transgenic plant cell according to one of claims 1 to 3, where the hexokinase is an unregulated enzyme. 6. Transgenic plants according to one of claims 1 to 5, where where the hexokinase is a glucokinase. 7. Transgenic plant cell according to one of claims 1 to 6, wherein the DNA sequence encoding the invertase from comes from a mushroom. 8. The transgenic plant cell according to claim 7, wherein the DNA Sequence encoding the invertase from Saccharomyces cerevisiae originates. 9. The transgenic plant cell according to claim 8, wherein the DNA Sequence encoding the invertase from the Suc2 gene Saccharomyces cerevisiae is.   10. Transgenic plant cell according to one of claims 1 to 9, the DNA sequence encoding the hexokinase comes from a prokaryotic organism. 11. The transgenic plant cell according to claim 10, wherein the DNA Sequence encoded a Zymomonas mobilis glucokinase. 12. Transgenic plant cell according to one of claims 1 to 11, the DNA sequence encoding the invertase under the control of a tissue-specific promoter, of a promoter leading to a particular development when the plant is active, or a promoter, which is inducible by external factors. 13. Transgenic plant cell according to one of claims 1 to 12, the DNA sequence encoding the hexokinase under the control of a tissue-specific promoter, of a promoter leading to a particular development when the plant is active, or a promoter, which is inducible by external factors. 14. Transgenic plant cell according to one of claims 1 to 13, which is a cell of a crop. 15. Transgenic plant cell according to claim 14, which is a cell an oil-storing plant. 16. Transgenic plant cell according to claim 15, which is a cell a rapeseed, soybean, sunflower or oil palm plant is. 17. The transgenic plant cell according to claim 14, which is a cell a starch-storing plant. 18. Transgenic plant cell according to claim 17, which is a cell a corn, rice, wheat, barley, rye, oat or Potato plant is.   19. Transgenic plant obtainable by regeneration Plant cell according to one of claims 1 to 18. 20. Transgenic plant containing plant cells after a of claims 1 to 18. 21. Transgenic plant according to claim 19 or 20, in which due to the increase in the rate of glycolysis in escort cells the transport rate of photoassimilates is increased. 22. Transgenic plant according to claim 19 or 20, in which due to the increase in the rate of glycolysis in cells of the Root epidermis or root hair the absorption of Minerals from the soil is elevated. 23. Transgenic plant according to claim 19 or 20, in which due to the increased rate of glycolysis in the endosperm and / or in the cotyledons of seeds the fatty acid bio synthesis is increased. 24. Transgenic plant according to claim 19 or 20, in which due to the increased rate of glycolysis in an organ of Fatty acid content is increased with a simultaneous reduction starch content. 25. Process for the production of transgenic plant cells with increased glycolysis rate, in which in vegetable cell len DNA sequences are introduced that a cytosoli encode invertase, as well as DNA sequences that a encode cytosolic hexokinase, and these DNA sequences zen expressed in the transformed plant cells will. 26. The method of claim 25, wherein the invertase is a de is regulated or unregulated enzyme.   27. The method of claim 25 or 26, wherein the hexokinase is a deregulated or unregulated enzyme. 28. The method according to any one of claims 25 to 27, wherein the Hexokinase is a glucokinase. 29. Recombinant DNA molecule containing the following DNA sequences:

• (i) a DNA sequence encoding a cytosolic invertase in combination with DNA sequences ensuring transcription and translation in plant cells; and
• (ii) a DNA sequence that encodes a cytosolic hexokinase in combination with DNA sequences that ensure transcription and translation in plant cells.

30. Recombinant DNA molecule containing a DNA sequence, which is a protein with the enzymatic activity of a Encoded hexokinase, in combination with DNA sequences, which are the transcription and translation in vegetable Ensure cells. 31. A recombinant DNA molecule according to claim 29 or 30, where where the hexokinase is a glucokinase. 32. Use of DNA sequences encoding an invertase ren, for the production of transgenic plant cells, the compared to non-transformed plant cells have an increased glycolysis rate. 33. Use of DNA sequences that a hexokinase co dieren, for the production of transgenic plant cells, compared to non-transformed plant cells have an increased rate of glycolysis.   34. Use according to claim 33, wherein the hexokinase is a Is glucokinase.