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  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to uransgenic plant cells and plants with a higher rate of glycolysis compared to non-transformed plants.
• the increase in the rate of glycolysis is achieved by introducing and expressing a DNA sequence encoding a cytosolic invertase, preferably a deregulated or unregulated invertase, and a DNA sequence encoding a cytosolic hexokinase, preferably a deregulated or unregulated hexokinase encoded, in plant cells.
• the invention also relates to methods and recombinant DNA molecules for the production of transgenic plant cells and plants with an increased glycolysis rate, and to the use of DNA sequences which contain proteins with the enzymatic activity of an invertase or proteins with the enzymatic activity encode a hexokinase for the production of plants which have an increased rate of glycolysis.
• WO 91/19896 further describes that the increase in starch biosynthesis by overexpression of a deregulated enzyme of ADP-glucose pyrophosphorylase leads to an increased allocation of sucrose in potato tubers, which are stored there in the form of starch. While many of these applications deal with the steps which either lead to the formation of photoassimilates in leaves (cf. also EP 466 995) or with the formation of polymers such as starch or fructans in storage organs of transgenic plants (for example WO 94 / 04692), there are no promising approaches to date which describe which modifications have to be introduced in the primary metabolic pathways in order to achieve an increase in the rate of glycolysis.
• An increase in the rate of glycolysis is important, for example, for all those processes in the plant for which larger amounts of ATP are required. This applies, for example, to many transport processes across membranes that are driven by a membrane potential or a proton gradient that is generated by the activity of an H + -ATPase. Furthermore, an increase in the rate of glycolysis is important for growth in the meristem, the switch from vegetative to generative Growth, as well as for the synthesis of various storage materials, in particular oils.
• the invention is therefore based on the object of making available plant cells and plants with an increased glycolysis rate as well as processes and DNA molecules for their production.
• the present invention thus relates to transgenic plant cells with a glycolysis rate which is increased in comparison to non-transformed plant cells, in which it is due to the introduction and expression of DNA sequences which code for a cytosolic invertase, and of DNA sequences, encoding a cytosolic hexokinase leads to an increase in invertase and hexokinase activity.
• the transgenic cells can each contain one or more DNA sequences which encode an invertase or a hexokinase.
• An increased glycolysis rate means that the transgenic plant cells which have been transformed with DNA sequences which lead to the additional synthesis of an invertase and a hexokinase in the cytosol of the cells have an increased glycolysis rate, preferably a glycolysis rate, compared to non-transformed plant cells which is increased by at least 30%, in particular one which is increased by at least 50% to 100%, and in particular one which is increased by more than 100% to 200%, preferably by more than 300%.
• the determination of the glycolysis rate by determining the corresponding metabolic intermediates is described in detail in the examples.
• the increase in the glycolate can be determined, for example, by increasing the concentration of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, pyruvate or ATP.
• the increase in the rate of glycolysis is preferably determined by determining the increase in pyruvate.
• the increase in the rate of glycolysis is compared. . determined with non-transformed cells. This means that material from plants which were used as the starting material for the introduction of the above-mentioned DNA sequences is used to determine the glycolysis rate and the glycolysis rate determined in this is compared with that of plants corresponding type or line after the transformation with the DNA sequences described above.
• the provision of larger amounts of ATP through an increased glycolysis rate enables, for example, the increase in various energy-dependent transport and growth processes.
• the provision of higher pyruvate concentrations as a result of an increased glycolysis rate leads to increased amounts of AcetylCoA, which is used, for example, for the increased synthesis of oils, fats or isoprenoid derivatives can be. If DNA sequences which enable the synthesis of polyhydroxyalkanoic acids are simultaneously expressed in the cells, the acetylCoA can also be used to form such alkanoic acids.
• the DNA sequences which encode an invertase or a hexokinase are DNA sequences which encode an invertase or hexokinase, in comparison to invertases or hexoki which normally occur in plant cells ⁇ noses are deregulated or unregulated.
• Deregulated means that these enzymes are not regulated in the same way as the invertase and hexokinase enzymes normally formed in unmodified plant cells. In particular, these enzymes are subject to other regulatory mechanisms, ie they are not inhibited to the same extent by the inhibitors present in the plant cells or are allosterically regulated by metabolites.
• Deregulated preferably means that the enzymes have a higher activity than endogenously expressed invertases or hexokinases expressed in plant cells. Unregulated means in the context of this invention that the enzymes in plant len Zel ⁇ no regulation subject.
• the DNA sequences which encode a protein with the enzymatic activity of an invertase can be both DNA sequences which encode prokaryotic, in particular bacterial, invertases and those which eukaryotic, ie invertases from plants, algae, Coding mushrooms or animal organisms.
• fungi also includes yeasts, in particular those of the genus Saccharomyces, such as, for example, Saccharomyces cerivisiae.
• the enzymes encoded by the sequences can be either known enzymes occurring in nature which have different regulation by various substances, in particular by the plant invertase inhibitors, or enzymes which by mutagenesis of DNA sequences which encode known enzymes from bacteria, algae, fungi, animals or planters.
• the DNA sequences encode proteins with the enzymatic activity of an invertase from fungi.
• Such enzymes have the advantage that they are not regulated by plant invertase inhibitors in comparison to plant invertases.
• DNA sequences encoding an invertase from Saccharomyces are preferably used. Such sequences are known and described (see. Taussig et al., Nucleic Acids Res. 11 (1983), 1943-1954; EP-A2 0 442 592).
• 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 pimpinellifolium, Z12026 L. pimpinellifolium, X73601 A. sativa, S70040 acid invertase, V01311 yeast gene, U11033 Arabidopsis thaliana, X81795 B.
• the DNA sequences which encode a procyne with the enzymatic activity of a hexokinase can be both those which encode prokaryotic, in particular bacterial, invertases and those which encode eukaryotic invertases, i.e. encode those from plants, algae, fungi or animal organisms.
• Hexokinases (EC 2.7.1.1) are enzymes that catalyze the following reaction:
• the hexokinases encoded by the DNA sequences can be known, naturally occurring enzymes, which have different regulation by different substances, as well as enzymes, which by mutage ⁇ nese from DNA sequences that encode known enzymes from bacteria, algae, fungi, animals or plants.
• DNA sequences which encode enzymes with hexokinase activity have been described from a whole series of organisms, for example from Saccharomyces cerevisiae, humans, rats and various microorganisms (for the DNA sequences see: EMBL gene bank access numbers M92054, LO4480, M65140 , X61680, M14410, X66957, M75126, J05277, J03228, M68971, M86235, X63658).
• these are DNA sequences which code for glucokinases, in particular glucokinases, which are subject to reduced allosteric regulation, for example by glucose-6-phosphate.
• Glucokinases (EC 2.7.1.2) are hexokinases with a high affinity for glucose, which catalyze the following reaction: Glucose -r ATP ⁇ —> glucose-6-phosphate + ADP
• the DNA sequences encode a giucokinase from Zymomonas mobilis (Barneil et al., J. Bacteriol. 172 (1990), 7227-7240; EMBL gene bank access number M60615). Further glucokinases from humans and rats have been described (for the DNA sequences see: EMBL gene bank accession numbers M69051, M90299, J04218 and M25807).
• DNA sequences which encode an invertase or a hexokinase can be isolated from any organism with the aid of the already known DNA sequences mentioned above. Methods for the isolation and identification of such DNA sequences are known to the person skilled in the art, for example hybridization with known sequences or by polymerase chain reaction using primers which are derived from known sequences.
• the enzymes encoded by the identified DNA sequences are then examined for their enzyme activity and regulation. Methods for determining the invertase or hexokinase activities are familiar to the person skilled in the art.
• the regulatory properties of the proteins encoded by the DNA sequences can be changed further in order to obtain de-regulated or unregulated enzymes.
• the DNA sequences which encode a cytosolic invertase or hexokinase can in principle be under the control of any promoter which is functional in plant cells.
• the expression of said DNA sequences can generally take place in any tissue of a plant regenerated from a transformed plant cell according to the invention and at any time, but is preferably found in such tissues instead, in which an increased glycolysis rate is advantageous either for the growth of the plant, for the uptake and transport of ions and metabolites or for the formation of ingredients within the plant.
• Promoters which ensure specific expression in a specific tissue, at a specific development time of the plant or in a specific organ of the plant therefore appear to be particularly suitable.
• Promoters which are specifically active in the endosperm or in the cotyledons of seeds forming therefore appear to be particularly suitable for increasing the fatty acid biosynthesis as a result of an increased acetylCoA content in seeds of oil-forming plants such as oilseed rape, soybean, sunflower and oil palms.
• Such promoters are, for example, the Phaseolin promoter from Phaseolus vulgaris, the USP promoter from Vicia faba or the HMG promoter from wheat.
• promoters that ensure seed-specific expression.
• starch-storing plants e.g. This increases the rate of glycolysis in the seeds of maize, wheat, barley or other cereals, and there is an increased formation of pyruvate and acetylCoA and an increased fatty acid biosynthesis. This means that a change in the flow of photoassimilates from starch towards pyruvate-dependent biosynthetic pathways, e.g. fatty acid biosynthesis.
• promoters which are active in storage organs such as tubers or roots, e.g. in the root of the sugar beet or in the tuber of the potato.
• the expression of the DNA sequences encoding an invertase or hexokinase leads to a redirection of biosynthetic pathways in the sense of the formation of less sugar or starch and an increased formation of pyruvate and acetyl-CoA due to the increased rate of glycolysis.
• promoters which are specific at the time the flowering induction are activated or are active in tissues that are necessary for the flowering induction. Promoters can also be used which are controlled only by external influences. Be activated at the time, for example by light, temperature, chemical substances (see, for example, WO 93/07279).
• promoters are of interest, for example, which have root hair or root epidermis-specific expression.
• promoters are of interest, for example, which have a transmission cell-specific expression.
• Such promoters are known (for example the promoter of the rolC gene from Agrobacterium rhizogenes).
• DNA sequences which encode an invertase or hexokinase can, apart from a promoter, advantageously be linked to DNA sequences which ensure a further increase in transcription, for example so-called enhancer elements, or to DNA sequences, which are in the transcribed area and which ensure a more efficient translation of the synthesized RNA into the corresponding protein (so-called translation enhancer).
• enhancer elements DNA sequences which are in the transcribed area and which ensure a more efficient translation of the synthesized RNA into the corresponding protein
• translation enhancer a promoter
• Such regions can be obtained from viral genes or suitable plant genes or can be produced synthetically. They can be homologous or heterologous to the promoter used.
• the DNA sequences which code for an invertase or a hexokinase are linked to 3 1 - non-translated DNA sequences which ensure the termination of the transcription and the polyadenylation of the transcript.
• Such sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens. These sequences are interchangeable.
• the DNA sequences which encode an invertase or hexokinase are preferably stably integrated into the genome in the plant cells according to the invention.
• the transgenic plant cells which, owing to the additional expression of a cytosolic invertase and a cytosolic hexokinase, have an increased rate of glycolysis, can in principle be cells of any plant species.
• cells of monocotyledonous and dicotyledon plant species in particular cells of starch-storing, oil-storing or agricultural useful plants, such as, for example, rye, oats, barley, wheat, potato, corn, rice, rapeseed, peas, sugar beet, Soybean, tobacco, cotton, sunflower, oil palm, wine, tomato etc. or cells of ornamental plants.
• starch-storing, oil-storing or agricultural useful plants such as, for example, rye, oats, barley, wheat, potato, corn, rice, rapeseed, peas, sugar beet, Soybean, tobacco, cotton, sunflower, oil palm, wine, tomato etc. or cells of ornamental plants.
• the plant cells according to the invention can be distinguished from corresponding non-transformed plant cells in that they contain foreign DNA sequences which are stably integrated into the genome and which encode a cytosolic invertase or a cytosolic hexokinase.
• the term “foreign DNA sequence” means the following: on the one hand, it can be DNA sequences that are heterologous with respect to the transformed plant cell, i.e. do not naturally occur in such a plant cell. If the DNA sequences are those which naturally occur in the transformed plant cells, "foreign” means that they are integrated in the genome of the transformed plant cells at a location where they do not occur naturally. ie they are in a new genomic environment. This can be verified, for example, by a Southern blot analysis. Furthermore, the DNA molecules introduced into the plant cells are generally recombinant DNA molecules, i.e. molecules that are composed of different segments that do not occur naturally in this combination.
• the present invention furthermore relates to transgenic plants which contain transgenic plant cells according to the invention. hold. Such plants can be generated, for example, by regeneration from a plant cell according to the invention.
• the loading of the sieve element-condenser cell complex with sucrose can be increased by the sucrose-proton co-transporter, which leads to an increase in the transport rate of photoassimilates.
• the absorption of inorganic ions such as phosphate, sulfate, nitrate, etc. from the soil can be increased via the root.
• An increase in the rate of glycolysis specifically in root cells, in particular in root hairs and epidermal cells can lead to an increased excretion of protons into the soil due to the increased H + -ATPase activity.
• Such acidification of the soil leads to mobilization and thus easier absorption of various minerals, such as phosphate from the soil.
• AcetylCoA is also important for many other processes that occur naturally in plants, such as isoprenoid biosynthesis, but also for the formation of polymers such as polyhydroxyalkanoic acids (see, for example, Poivier et al., Bio / Technology 13 ( 1995), 142-150).
• the result is a reduction in the amount of starch in the corresponding tissue with a possible simultaneous increase in the fatty acid biosynthesis.
• the present invention relates to a method for producing transgenic plant cells which have an increased glycolysis rate in comparison to non-transformed plant cells, in which DNA sequences which encode a cytosolic invertase are introduced into plant cells, and also DNA sequences , which encode a cytosolic hexokinase, and these sequences are expressed in the transformed plant cells.
• Such a method preferably consists of the following steps:
• step (i) a promoter which ensures transcription in plant cells;
• the promoters, further flanking DNA sequences, and for the selection and modifications of the DNA sequences which encode an invertase or a hexokinase were carried out.
• the DNA sequences which encode an invertase or a hexokinase can either be located on separate DNA molecules or together on a recombinant DNA molecule. If the sequences are on two different DNA molecules, the transfer of the DNA molecules can either take place simultaneously or in such a way that plant cells are first transformed with a DNA molecule and then selected plant cells or plants are subsequently transformed with the second DNA molecule become. Furthermore, plants which express both an additional cytosolic invertase and an additional cytosolic hexokinase can be produced by first generating two independent transgenic plant lines which code for an invertase or a hexokinase and then crossing them.
• the transfer of the DNA molecules which contain DNA sequences which encode invertase or hexokinase is preferably carried out using plasmids, in particular those plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, with so binary binary plasmids or Ti plasmids of the Agrobacterium tumefaciens system.
• plasmids in particular those plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, with so binary binary plasmids or Ti plasmids of the Agrobacterium tumefaciens system.
• other systems for introducing DNA molecules into plant cells are also possible, such as the so-called biolistic method or the transformation of protoplasts (cf. Willmitzer L. (1993), Transgenic Plants, 3iotechnology 2; 627-659 for an overview). Basically, cells of all plant species are suitable for transformation.
• Both monocot and dicotyledonous plants are of interest. Transformation techniques have already been described for various monocot and dicot plant species.
• Cells of agricultural crops are preferably used in the processes, in particular of cereals, for example rye, oats, barley, wheat, potatoes, maize, rice, rapeseed, peas, sugar beet, soybeans, tobacco, cotton, sunflower, oil palm, wine , Tomato etc. or cells of ornamental plants.
• the invention also relates to the transgenic plant cells obtainable from the process and plants obtainable therefrom by regeneration, which have an increase in the rate of glycolysis due to the additional expression of a cytosolic invertase and a cytosolic hexokinase.
• the present invention relates to propagation material of plants according to the invention which contains cells according to the invention.
• This can be any type of tissue or organ of the plants according to the invention which enables the propagation.
• These include, for example, tissue cultures of cells according to the invention, seeds, fruits, rhizomes, cuttings, seedlings, tubers etc.
• the present invention further relates to recombinant DNA molecules which contain a DNA sequence which codes a protein with the enzymatic activity of a hexokinase, preferably a glucokinase, in combination with DNA sequences which transcription and translation in ensure plant cells.
• a hexokinase is preferably a deregulated or unregulated enzyme.
• the present invention further relates to recombinant DNA molecules which comprise the following DNA sequences:
• the present invention relates to the use of DNA sequences which encode an invertase, preferably a deregulated or unregulated, for the production of transgenic plant cells which have an increased glycolyzerate in comparison to non-transformed plant cells.
• the invention also relates to the use of DNA sequences which encode a hexokinase, preferably a deregulated or unregulated, for the production of transgenic plant cells which have an increased glycolytic rate in comparison to non-transformed plant cells. Description of the picture
• Figure 1 shows the 12.68 kb plasmid pB33Hyg-GK.
• the plasmid contains the following fragments:
• Fragment A contains the Dral-Dral fragment (position -1512 to position +14) of the promoter region of the patatin gene 333 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).
• fragment B contains a DNA fragment with the coding region of the 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.
• the vector pUC18 was used for cioning in E. coli.
• the gene constructions were cloned into the binary vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66 (1990), 221-233).
• E. coli strain DH5 ⁇ (Bethesda Research Laboratories, Gaithersburgh, USA) was used for the pUC vectors and for the pBinAR constructs.
• the DNA was transferred by direct transformation using the Höfgen and Willmitzer method (Nucleic Acids Res. 16 (1988), 9877).
• the plasmid DNA of transformed Agrobacteria was isolated by the method of Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523) and analyzed by gel electrophoresis after a suitable restriction cleavage.
• the starch content and the dry substance of the potato tuber were determined by means of the determination of the specific weight (Scheele et al., Landw. Vers. Sta. 127 (1937), 67-96) according to the following formulas:
• the phosphoryiated intermediates were determined on a two-wavelength photometer (Sigma ZWS 11) by means of coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology 174, 518-552).
• the reaction buffer contained: 50 mM Hepes-KOH pH 7.0;
• the measurement is carried out at 25 ° C. with 50 to 100 ⁇ l extract.
• the reaction buffer contained: 200 mM glycine pH 8.7;
• G6P glucose-6-phosphate
• F5P Fmctose-6-phosphate
• G1P glucose-1-phosphate
• the reaction buffer contained: 50 mM Hepes-KOH pH 7.0;
• the reaction buffer contained: 50 mM Hepes-KOH pH 7.0;
• the reaction buffer contained: 50 mM Hepes-KOH pH 7.0;
• the reaction buffer contained: 100 mM Tris-HCl pH 8.1;
• Glucokinase and fructokinase activity was determined according to Renz et al. (Planta 190 (1993), 156-165), the sucrose synthase and invertase activity according to Zrenner et al. (Plant J. 7 (1995), 97-107), the phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, pvruvate kinase activity according to Burell et al. (Planta 194 (1994), 95-101), and the phosphoglucomutase activity according to Pressey (Journal of Food Science 32 (1967), 381-385).
• Tubers (approximately 30 g) were taken directly from plants from the greenhouse and placed in an infrared gas analyzer (Binos 100 Rosemound, Dusseldorf, FRG) within 30 minutes. The CO 2 production was determined over 20 minutes at 20 ° C. and evaluated with the software from Waltz (Effeltrich, FRG).
• the coding region of the glucokinase gene from Zymononas mobilis was amplified using the polymerase chain reaction (PCR) starting from genomic Zymomonas mobilis DNA.
• the sequence of the Zymomonas mobilis glucokinase is entered in the GenEMBL database with the Accessicn number M60615.
• the amplified fragment corresponds to the region from nucleic acids 5128 to 6153 of this sequence.
• An Asp718 interface was inserted at the 5 'end and a HindIII interface at the 3' end.
• the 1025 bp PCR fragment was cloned into the vector pUCBM20 via the two additional interfaces.
• the entire coding region of the glucokinase was subcloned into the vector pBluescriptSK after restriction digestion with EcoRI and HindIII.
• extracts from ⁇ . coli cells containing the resulting plasmid pSK-GK were found to have a 100-fold increase in glucokinase activity compared to extracts from untransformed E. coli cells.
• the cells of a 20 ml overnight culture were harvested and resuspended in 500 ⁇ l extraction buffer (30 mM KH 2 PO 4 ; 2 mM MgCl 2 ; 10 mM 2-mercaptoetha- nol; 0.1% (Vol. / Vol.) Nonid ⁇ t? 40).
• the suspension was mixed vigorously four times for 30 seconds. After centrifugation, the glucokinase activity in the cell-free extract was carried out as in Scopes et al. (Biochem. J. 228 (1985), 627-634).
• the insert was recloned into a binary vector derived from pBIN19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8720).
• the following plasmid was created: the plasmid pB33Hyg-GK (cf. FIG. 1).
• the construct contains the B33 promoter from Solanum tuberosum (Rocha-Sosa et al., ⁇ MBO J. 8 (1989), 23-29).
• the construct pB33Hyg-GK was created as follows: Since the construct was to be used for the transformation of already transgenic potato plants which express the NPT-II gene, the plasmid pBIB, which codes for the HPT gene, was used which contains hygromycin B phosphotransferase (Becker, Nucl. Acids Res. 18 (1990), 203). The promoter of the B33 gene from Solanum tuberosum was inserted as a Dral fragment (position -1512 to +14 according to Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) into the Sacl Cloned the plasmid pUC19.
• the promoter region was cloned into the binary vector pBIN19, which contains the termination signal of the octopine synthase gene from Agrobacterium tumefaciens in the direct vicinity of a polylinker from M13mpl9. This resulted in pB33.
• the promoter polylinker terminator fragment of plasmid pB33 was cloned as an EcoRI / HindIII fragment into the plasmid pBIB linearized with EcoRI and HindIII. This resulted in the plasmid pB33Hyg.
• the coding region of the glucokinase was then isolated after Asp718 / Sall digestion of the plasmid pSK-GK and Asp718 / Sall was cloned into the plasmid pB33Hyg. This resulted in the plasmid pB33Hyg-GK, which is used for the transforma- tion of the transgenic potato line U-Inv-2 (line 30) was used.
• the binary plasmid is introduced into the cells by direct transformation according to the method of Höfgen & Willmitzer (Nucl. Acids Res. 16 (1988), 9877).
• the plasmid DNA of transformed agrobacteria was determined by the method of Birnboim et al. (Nucl. Acids Res. 7 (1979), 1513-1523) isolated and analyzed by gel electrophoresis after suitable restriction cleavage.
• the enzyme activities shown here are the average of at least five measurements based on five independent plants.
• GK-41, GK-29 and GK-38 were amplified and 15 plants in each case were transferred to a greenhouse.
• the tubers were harvested 4 months later.
• soluble sugars such as glucose, fructose and sucrose surprisingly showed that the seven-fold increase in the glucose concentration in the U-Inv-2 plants compared with the wild type is greatly reduced by the expression of the glucokinase, so that the amount of glucose only is still 30% of the amount of glucose in tubers of WT control plants.
• the fructose concentration in the transgenic lines has not changed compared to the control plants.
• the strong reduction in the amount of sucrose in the U-Inv-2 plants is partially offset by the expression of the glucokinase (cf. Table III).
• the tubers of the GK-38 plants only contain 40% starch and 50% soluble sugar compared to tubers from untransformed control plants.
• 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.
• 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 " mg fresh weight.
• the values shown here are the mean values of six measurements based on six independent plants. The values are given in mmol C0 2 g fresh weight.
• the controls given in the experiments described above are each non-transformed plants of the plant species or subspecies used for the transformation.

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