HU221515B - Transgenic plants with improved biomass production - Google Patents

Abstract

In the present invention, transgenic plant cells are described in which easily mobilizable phosphate pools are produced outside the avacuolum due to the introduction and expression of specific DNA molecules. Transgenic plants containing such cells can be characterized by increased biomass production and / or altered flowering properties. In addition, processes are described that provide improved yield and / or modified flowering properties. ŕ

Description

The present invention relates to transgenic plant cells in which delivery and expression of specific DNA molecules results in the formation of easily mobilizable phosphate pools outside the vacuole. Transgenic plants containing such plant cells exhibit increased biomass production and / or modified flowering compared to untransformed plants.

The present invention further relates to methods for increasing yield or modifying the flowering properties of a plant, wherein the plants are modified such that easily mobilizable phosphate pools are formed outside the vacuole.

Efforts to increase crop yields are needed to provide food for the world's growing population

In recent decades, it has been possible to continuously increase the yield of cultivated crops in the agricultural sector due to industrialization. In particular, the mechanization of agriculture, modernized crop production, plant protection and the provision of advanced nutrients using different fertilizers of plants contributed to this.

Currently, the improvement of the above-mentioned instruments is hardly expected to increase returns. For example, in a biosphere that restricts plant growth, the supply of nutrients such as nitrogen and phosphorus to plants is currently generally solved. This means that further fertilization does not lead to increased yields. Developing new crop varieties with higher yields using crop breeding methods is time consuming & negative tufts of the resulting crop varieties *. for example, lower reactivity to diseases is often tolerated by increased use of pesticides.

The possibility of producing higher yielding plant species is through specific genetic engineering of plants.

For example, the expression of prokaryotic asparagine synthase in plant cells, which induces an increase in biomass production of plants, is described (EP-B10 511979).

In other processes, attempts are made to modify the expression of genes that are involved in the synthesis pathway of storage materials or to modify the distribution of photosyntheses produced by photosynthesis by preferentially supplying the collimating organs, particularly storage organs, with the photosynthesis assimilate. In this process, we either try to improve the ability of the "collecting" organs to absorb the appropriate form of transport of photoassimilates, usually sucrose, or to directly influence the transport.

For example, EP 0 442 592 describes the expression of an invertase in potato, resulting in modified yields in such modified plants.

(Riesmeier et al., EMBO J. 13 (1994),

-7) also utilizing sucrose carrier for modifying sucrose transport (see WO 94/00574).

Thus, the problem of the present invention is to provide further genetic engineering methods which are generally applicable to plants to increase biomass production and / or yield.

This problem is solved by providing the embodiments described in the claims. Surprisingly, it has been found that the introduction and expression of DNA molecules encoding enzymatic activity proteins in transgenic plant cells leading to the production of phosphate pools outside the vacuole results in increased biomass production and thereby an increased yield of plants containing such cells. In addition, such plants have altered flowering properties.

Thus, the present invention relates to transgenic plant cells in which at least one readily mobilizable phosphate pool is formed by introducing and expressing a DNA molecule that encodes a protein involved in the synthesis of a phosphate-containing molecule outside the vacuole.

In the context of the present invention, increased biomass production of the plant results in increased biomass (measured as dry weight) of the entire plant and / or individual parts relative to the wild-type plant, preferably at least 5% and more preferably more than 10%. growth.

Thus, the production of abrasive seeds includes the increase in yields of parts of the agricultural stomach-bellowing plant, such as storage organs such as potato tubers, beets, seeds, fruits, leaves, stems, etc. growth in plants where phosphate pools are produced outside the vacuole relative to wild-type plants, preferably at least 5%, more preferably greater than 10%.

In general, such increases exceed those achieved by conventional cultivation methods

In the context of the present invention, flowering property, i.e., early flowering, is understood to mean that the transformed plants bloom at least a few days, preferably one to several weeks, in particular 1-2 weeks, in comparison to the untransformed plants.

In the context of the present invention, the term phosphate pool is understood to mean phosphate-containing molecules containing a covalently bonded phosphate atom from which the phosphate can be easily mobilized, i.e. released or transported to other molecules, by generally reversible enzymatic reactions. Thus, the availability of phosphate in cells for various phosphate-dependent reactions is increased. By "readily mobilizable" is meant that phosphate is readily available in the cytosol of the cell than is possible through the transport of phosphate from the vacuole to the cytosol. In the case of phosphate deficiency, the release of phosphorus from the vacuole to the cytosol generally takes several hours (Woodrow et al., Planta 161 (1984), 525-530).

Phosphate pools generally refer to pools of phosphate-containing molecules that do not interfere with cytosolic homeostasis, including the concentration of phosphate required for metabolic processes, but

From which the phosphate is readily liberated or delivered to other molecules, providing a means of guaranteeing an increased availability of phosphate. Particularly useful for this purpose are phosphate-containing compounds which do not occur in higher plants. Examples of such compounds are polyphosphate (Wood, Ann. Rev. Biochem. 57: 235-260 (1988)), a compound synthesized by most organisms, except for higher plants, as a phosphate storage agent, and acetyl phosphate, which in bacteria. It has been used to regenerate ATP (see, e.g., Matsuyama et al., J. Bactetiol. 171 (1989), 577-580).

According to the present invention, the phosphate pools may also contain trehalose-6-phosphate, phosphoenol pyruvate or fructose-1-phosphate.

Preferably, such phosphate pools outside of the vacuole are produced by introducing DNA molecules into plant cells that produce proteins whose enzymatic activity in the transgenic plant cells leads to the production of the corresponding phosphate pools and expressing these DNA molecules.

In a preferred embodiment, the DNA molecules introduced into plant cells encode proteins that have the enzymatic properties of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenol pyruvate mutase or a ketohexo kinase.

Polyphosphate kinases (ATP: polyphosphate phosphotransferase; E.C. 2.7.41) catalyze the following reaction: Polyphosphate + ATP Polyphosphate + 1 + ADP

The product of this reversible reaction is a linear polymer of phosphate residues. The lengths of the polymers can range from 3 to 1000 phosphate residues. The enzyme has already been described in several organisms, inter alpha in E. coli (Ahn and Komberg, J. Bioi. Chem. 265 (1990), 11734-11739; Akiyama et al., J. Bioi. Chem. 267 (1992)). 22556-22561), Klebsiella aerogenes (Kató et al., Gene 137 (1993) 237-242), S. cerevisiae (Felter and Stahl, Biochimie 55 (1973) 245-251), Propionibacterium shermanii. baa (Robinson and Wood, J. Bioi. Chem. 261 (1986), 4481-4485; Robinson et al., J. Bioi. Chem. 262 (1987), 5216-5222), Corynebacterium xerosis-bttn (Muhammed, Biochim). Biophys. Acta 54 (1961), 121-132) and Arthobacterium atrocyaneus (Levinson et al., J. Gen. Microbiol. 88 (1975), 65-74).

Acetate kinases (ATP: Acetate-Phosphotransferase; E.C. 2.7.2.1) catalyze the following reaction:

Acetate + ATP Acetylphosphate + ADP

The enzyme has been identified in many microorganisms and DNA sequences encoding acetate kinases have been isolated, such as the ack gene from E. coli strain K-12 (Matsuyama et al., 1989, J. Bacteriol. 171, 577-580) and Methanosarcina thermophila. ack gene (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829).

Phosphotransferase acetases (acetyl-CoA: orthophosphate acetyl transferase; E.C. 2.3.1.8) catalyze the following reaction:

Acetyl Coenzyme A + Orthophosphate Acetyl Phosphate + Coenzyme A

The DNA sequence encoding this enzyme has also been described, for example, the pta gene from Methanosarcina thermophila (Latimer and Light, J. Bacteriol. 175 (1993), 6822-6829).

Trehalose-6-phosphate synthase catalyzes the synthesis of trahalose-6-phosphate. Such an enzyme from yeast is described in U.S. Patent No. 5,422,254.

Phosphoenol pyruvate mutates catalyzes the synthesis of phosphono pyruvate. Such an enzyme from Tetrahymena pyriformis has been described, for example, by Seidel et al., Biochemistry 31 (1992), 2598-2608.

Ketohexokinase catalyzes the synthesis of fructose-1-phosphate. On a single cozumcM in® le spindle from rats and humans, νοΜκκοδ es gmfftrangsai (Biochem. J. 291 (1993), 179-186) and Bonthron et al. (Hûm. Mol. Genet 3 (1994) 1627-1631).

DNA molecules encoding proteins that lead to the production of phosphate pools outside of the vacuole, in particular proteins that have a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trenaiosose-tosylate synthase, a tostoenol-silyl niutase, or a ketohexone kinase enzyme. , may be genomic or cDNA molecules derived from any organism, preferably prokaryotic DNA molecules, in particular those that are lamentable to a bacterial organism. DNA molecules can be isolated by conventional molecular biological methods or synthesized. The molecules may further be modified by methods known to those of ordinary skill in the art to include plant-specific codons for enhancing expression in plant cells.

Preferably, DNA molecules that encode proteins with enzymatic properties of a polyphosphate kinase and exhibit low Kg, high ADP and high Kg value against ATP are used for delivery and expression in plant cells.

The activity of these enzymes is generally affected by the ATP: ADP ratio. This means that the phosphate pool is only newly produced when ATP is present in excess of ADP. This leads to depletion of the cytosolic phosphate concentration under favorable conditions for ATP production and to continuous phosphate reflux from the vacuum. These newly created pools have the advantage of being much easier to mobilize compared to the vacuole phosphate pool. In phosphate depletion in the cytosol, phosphate is usually released from the vacuum into the cytosol for several hours (Woodrow et al., Planta

HU 221 515 Bl

161 (1984), 525-530). Specifically, this means that such compounds foreign to plants are relatively rapidly degraded due to the enzymatic properties of the enzymes described above, and thus the phosphate-mobilized homeAtP: ADP ratio is reduced.

In another preferred embodiment, the DNA molecules introduced into plant cells and encoding a polyphosphate kinase are DNA molecules from E. coli, Klebsiella aerogenes, Neisseria meningitidis, or Synechocystia heads. Especially preferred is the ppk gene from E. coli (Akiyama et al., J. Biol. Chem. 267 (1992), 22556-22 561), the ppk gene from Klebsiella aerogenes-bfA (Kató et al., Gene 137 (1993), 237-242) from Neisseria meningitidis deposited in the GenBank and available under catalog number U16262. (Tinsley and Gottschlich; 1995), or Synechocystia sp., Deposited in the GenBank and available under accession number D64005. Sequence from strain PCC6803 (Kaneko et al., 1995).

In another preferred embodiment, DNA molecules encoding an acetate kinase derived from microorganisms Methanosarcina thermophila, E. coli, Haemophilus influenza, Bacillus subtilis or Mycoplasma genitalium are provided. Particularly preferred are the ack gene from K coli strain K-12 (Matsuyama et al., 1989, J. Bacteriol. 171, 577-580), the ack gene from Methanosarcina thermophila, and the sequence from Haemophilus influenza (Fleischmann et al., Science 269 (1995) 496-512), the sequence from Bacillus subtilis (Grundy et al., J. Bacteriol. 175 (1993) 7348-7355) or the sequence from Mycoplasma genitalium (Fraser et al. al., Science 270 (1995) 397-403). The DNA molecules encoding the phosphotransacetylase are preferably derived from Methanosarcina thermophila, Escherichia coli or Mycoplasma genitalium microorganisms. Especially preferred is the pta gene from Methanosarcina thermophila (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829), from E. coli described by Kakuda et al., J. Biochem. 1994), 916-922) DNA molecule or Matsuyama et al. (Biochim. Biophys. Acta (1994) 559-562) or a DNA molecule derived from Mycoplasma genitalium, which shows the sequence described by Fraser et al., Science 270 (1995) 397-403.

Preferably, the DNA molecules encoding the trichalose-6-phosphate synthase are from yeast. Preferably, the DNA molecule described in U.S. Patent No. 5,422,254,254 is used. Preferably, the DNA molecules encoding the phosphoenol pyruvate mutase are derived from Tetrahymena pyriformis, such as those described by Seidel et al., Supra.

Preferably, the DNA molecules encoding the ketohexokinase are human or rat, such as those described by Bonthron et al., Supra, or Donaldson et al., Supra.

In a further preferred embodiment, the DNA molecules encoding the protein leading to polyphosphate synthesis can be introduced into plant cells at the same time as the DNA molecules encoding the protein having the enzymatic activity of acetate kinase and the DNA sequence having the enzymatic activity of phosphotransacetylase.

The enzymatic activity of acetate kinase and phosphotransacetylase leads to the conversion of acetyl coenzyme A and ADP to acetate and ATP. The resulting ATP can then be used by polyphosphate kinase for the synthesis of polyphosphate.

The mobilization of phosphate pools produced by the expressed proteins in cells outside the vacuole, in particular the phosphate mobilization, can be influenced by other enzymes in addition to the phthalimides catalyzing the reversible reactions described above. Enzymes using polyphosphate as substrates are also known. In this case, DNA sequences encoding such enzymes must be introduced into plant cells and expressed. Known is, for example, polyphosphate glucokinase, which has already been described in many microorganisms, such as Mycobacterium phlei (Szymona, Bull. Acad. Pol. Sci. Ser. Sci. Bioi. 5 (1956), 379-381; Szymona and Ostrowski, Biochim. Biophys. Acta 85 (1964) 283-295), in Propionibacterium shermanii (Pepin and Wood, J. Bioi. Chem. 261). (1986), 4476-4480), Corynebacterium xerosis baa (Dirheimer and Ebei, Bull. Soc. Chim. Biol. 50 (1968), 1933-1947) and Mycobacterium tuberculosis baa (Szymona et al., Acta Biochim). 24: 133-42 (1977). Other enrimpk that use polyphosphate as substrate and which are therefore useful for the mobilization of pnHl-asphite include: polyphosphate: AMP-phosphotransferase (Bonting et al., J. Bacteriol. 173 (1991) 6484-6488; Dirheimer and Ebei). Sci. Paris 260 (1965), 3787-3790), 1,3-diphosphoglycerate: polyphosphate phosphotransferase (Kulaev et al., Biokhimiya 33 (1968) 419-434; Wood and Goss, Proc. Natl. Acad. Sci. USA 82 (1985), 312-315), polyphosphate-dependent AD kinase (Murata et al., Agric. Bioi. Chem. 44 (1980), 61-68), exopolyphosphetase (see, for example, Akiyama et al., J. (Biol. Chem. 268 (1993), 633-639; Wurst and Komberg, J. Biol. Chem. 269 (1994), 10996-11001) and endopolyphosphatase (EC 3.6.1.10; see, for example, Kowalczyk and Phillips, 1978). Analyt Biochem 212 (1993) 194-205).

Essentially, it is possible that the protein expressed in the transgenic plant sqt, due to its enzymatic activity, leads to the extracellular production of phosphate pool, especially polyphosphate or acetyl phosphate, can be localized in any constituent of the plant cell. To achieve a specific localization, the coding region must be linked to DNA sequences that ensure localization in the appropriate component. Preferably, the protein to be expressed is localized to the cytosol, plastids, or mitochondria. No specific signal is required for localization in the cytosol. When using prokaryotic DNA molecules, it should be noted that the coding region does not show signal sequences that express exp4.

For example, signal sequences that guarantee localization in mitochondria or plastids are known (see, e.g., Braun et al., 1992, EMBO J. 11, 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85: 846-850 (1988).

The plant cells into which the above described

The DNA molecules supplied are generally derived from any plant, especially any monocotyledonous or dicotyledonous plant. We prefer to use plant cells derived from agriculturally useful plants, especially cereals (e.g., barley, rye, oats, millet, rice, maize, wheat, etc.), fruitCSQ & sTOl, (from WgCMQwOi, FepCeDOl, CUKOITcpSOOl, scyl, szcy & , beans, tomatoes, etc.) or ornamental plants. Photosynthetically active cells are particularly preferred.

The present invention relates to transgenic plants comprising the transgenic plant cells of the present invention. These can be obtained, for example, by regeneration of the transgenic plant cell described by methods known to those skilled in the art of sjalcembg.

It has been found that, compared to untransformed (wild-type) plants, such plants are characterized by increased biomass isolation and modified flowering properties, especially early flowering and early flowering.

The present invention also relates to propagation material of the plant of the invention, e.g. seeds, fruits, tubers, cuttings and rootstocks, etc.

The present invention further relates to methods for enhancing or enhancing yields and / or improving the flowering properties of a plant, which is characterized by the ability to easily mobilize phosphate pools in plant cells, iOsZaK XOkCmSOu CxCIiHaDSGgSC fnZEOnya a SGfi & k. & 523Ι1Π8Γ.

Typically, such a procedure involves the following steps:

(a) Production of an expression cassette containing the following DNA sequences (i) a plant cell-operable promoter that provides transcription of a case-following DNA sequence;

(ii) at least one DNA sequence encoding a protein whose enzymatic activity results in an easily mobilizable phosphate pool production outside the vacuole and which binds to the 3 'end of the promoter in sense orientation; and (iii) optionally adding a termination signal to terminate transcription and adding a polyA tail to the resulting transcripts that bind to the 3 'end of the coding region;

(b) transforming the plant cells with the expression cassette generated in step (a) and integrating the expression cassette into the plant genome in a stable manner; and (c) regenerating complete intact plants from the transformed plant cells.

The promoter mentioned in step (i) of the process of the present invention can be essentially any plant-based promoter. Expression of said DNA sequences can generally occur at any time in any tissue of the transformed plant. Suitable promoters include, for example, the cauliflower mosaic virus (Odell et al., Naturre 313 (1985) 810-812), a 35S promoter that is constitutive in all tissues of the plant. expression. However, promoters which lead to the expression of sequences in a specific tissue of the plant, preferably in the photosynthetically active tissue, are also used, see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245-2251) or only externally. stimuli at a given time (see, for example, WO / 9307279).

For the DNA sequence referred to in (ii), any DNA molecule described for transgenic cells may be suitable.

The purpose of the terminating sequence is to correctly terminate transcription and to add a poly-A tail to the transcripts, which are believed to play a role in stabilizing the transcripts. Such elements have been described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged arbitrarily.

The present invention relates to expression cassette expression. ten are preferably located on a plasmid, preferably p35S-PPK (Fig. 1) or p35S-ACK (Fig. 3), and preferably introduced into a plant cell using a plasmid suitable for transformation of plant cells. In the examples of the present invention, the pBinAR binary vector (Hofgen and Willmitzer, Plant Sci. 66 (1990), 221-230) is used. This vector is a derivative of the binary vector pBin19 (Bevan, Nucl. Acids Sl 12 (1984) 8711-8721), commercially available (Clontech Laboratories, Inc., USA). However, any other plant transformation vector that incorporates an expression cassette into the expression gene and plant genome can also be used.

With the help of the expression cassette constructed in step (a), the cells of any plant can be used for the transformation according to step (b), preferably monocotyledonous or dicotyledonous plants. In the process, we prefer to use crops useful for agriculture, particularly cereals (such as barley, rye, oats, millet, rice, corn, wheat, etc.), fruits, potatoes, rape, sugar beet, soybeans, vegetables (such as peas, beans, tomatoes, etc.) or ornamental plants.

The present invention also relates to transgenic plant cells and transgenic plants obtained from the process of the invention. Transgenic plants are characterized by expressing in their genome a protein in the cells of these plants by incorporation and stable integration into the genome of the expression cassette constructed according to the method of the present invention, which by its enzymatic activity acts on polyphosphate, acetyl phosphate, trehalose-6-phosphate. extraction of phosphoenol pyruvate or fructose-1-phosphate outside the vacuum.

HU 221 515 Β1

The present invention specifically relates to a transgenic plant comprising a DNA sequence encoding a protein having at least one polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphoenol pyruvate mutase, or a protein sequence encoding a protein having enzymatic activity of ketohexokinase. binds to regulatory DNA regions for transcription and translation, is stably integrated in the genome and polypeptide, acetyl phosphate, trehalose-6-phosphate, phosphoenol pyruvate or fructose-1-phosphate is produced in plant cells outside the vacuole. expression.

Another object of the present invention is to provide DNA molecules encoding proteins which, by virtue of their WBMwqtibtS alrtivitamiknalf activity, exert their action on readily mobilizable phosphate pookdu, particularly polyphosphate, acetyl phosphate, trehalose-6-phosphate, fructose-1-phosphate or for the production of pyruvate in plant cells outside the vacuole, for transformation of plant cells and for expression in plant cells, in particular for the development of the phenotype of these cells. The present invention relates specifically to the use of DNA molecules which encode a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trehalose-6-phosphate synthase, a phosphatol pyruvate mutase or a protein having enzymatic activity in plant cells, and the polyphosphate. , trehalose-6-phosphate, phosphoenol pyruvate or fructose-phosphate in plant cells outside the vacuole. Examples of such DNA molecules include, in particular, the DNA molecules described above, which encode proteins having the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a trichalose-6-phosphate synthase, a phosphoenol pyruvate mutase or a ketohexokinase.

For the preparation of foreign genes into higher plants, there are several cloning vectors containing the replication signal for E. Coli and a marker gene for selection of transformed bacterial cells. Examples of such vectors include pBR322, pUC series, M13 series, pACYC184 vectors, and the like. The desired sequence can be introduced into the vector at a convenient restriction site. The resulting plasmid was used to transform E. coli cells. The transformed £. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid was swallowed. The analysis method used for the DNA characterization of the recovered plasmid is generally restriction analysis, gel electrophoresis and other biochemical molecular biological methods. At each manipulation, the plasmid DNA is cleaved and the resulting DNA fragments can be linked to other DNA sequences. Each plasmid DNA sequence may be cloned into the same or different plasmids. There are several techniques available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells by T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes for transformation, protoplast fusion, injection, DNA electroporation, biology delivery techniques, and others.

There are essentially no specific requirements for the plasmid used for injection of DNA into plant cells and electropolishing. Simple plasmids such as pUC-derived plasmids may also be used. However, if the entire plant is to be regenerated from such transformed plants, the presence of a selectable marker gene is required. The desired gbask. additional DNA sequences may be required depending on the method of delivery to plant cells. For example, when Ti or Ri is used to transform plant cells, at least the right border, but often the right and left borders of the Ti and Ri plasmids, must be linked as adjacent regions to the genes to be introduced.

If agro-htertrins are used for transformation, the DNA to be introduced must be cloned into specific plasmids, either into an intermediate vector or into a binary vector. Due to sequences homologous to those in the T-DNA, the intermediate vectors can be integrated into the Ti or Ri plasmids of the agrobacteria by homologous recombination. Bz also contains a vir region required for T-DNA transfer. Intermediate vectors are unable to replicate in the agmhalrt ^ rinmolchan Using a plasmid hafpnr, the intermediate vector can be introduced into Agrobacterium ttaH ^ hriens (conjugation). The binary vectors are £. coli and agrobacteria. These contain a selectable marker and a binding or polyhedron flanked by the right and left regions of the T-DNA border. These can be directly transformed into agrobacteria (Holsters et al., Mol. Gene. Genet. 163 (1978), 181-187). The host agrobacteria must also include a plasmid containing a vir region. The vir region is required for the transfer of T-DNA into plant cells. Additional T-DNA May Be Presented The agrobacteria thus transformed are used to transform plant cells.

The use of T-DNA for transformation of plant scats has been extensively studied and is well described in EP 120516; Hoekema, In: The Binary Plant Vector System Offsetdmkkerij Kanters B.V., Alblasserdam, Chapter V; Fraley et al., Crit. Port. Plant. Sci., 4 (1985), 1-46 and An et al., EMBO J. 4 (1985), 277-287.

It is advisable to co-cultivate plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA to a plant cell. Whole plants from infected plant material (e.g., leaf explants, stem segments, roots, and protoplasts or cultured plant cell suspensions) may be regenerated in appropriate medium containing antibiotics or biocides to select for transformed cells. Plants obtained in this manner can be assayed for the presence of introduced DNA.

When the introduced DNA is integrated into the plant cell genome, it is generally stable in the genome and originally

HU 221,515 B1 remains in the progeny of the transformed cell. In general, it contains a selectable marker that confers resistance to transformed plant cells with biocides or antibiotics such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin, etc.

The individually selected marker therefore allows the selection of transformed cells from cells lacking the introduced DNA.

Transformed cells grow normally within the plant (see McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be normally propagated and crossed with plants having the same transformed hereditary factor or other hereditary vector. The resulting hybrid individuals possess the appropriate phenotypic properties

Two or more generations must be cultivated to ensure that the phenotypic trait remains stable and is inherited. The seeds should be collected to ensure that the appropriate phenotype or other species is maintained

Media and solutions used:

20xSSC 175.3 g NaCl g sodium citrate

Make up to 1000 ml with ddH ₂ O and adjust to pH 7.0 with 10 N NaOH

ΙΟχΜΕΝ 200mMMOPS mM sodium vinegar in 10mM EDTA pH 7.0

NSEB Buffer 0.25 M Sodium Phosphate Buffer pH 7.2 7% SDS ImM EDTA 1% ΒΣΑ (w / v)

Description of the figures:

Figure 1 shows plasmid p35S-PPK

Structure of the plasmid:

A = Fragment A: CaMV 35 S promoter, nucleotides 6909-7437 (Franck et al., Cell 21 (1980) 285-294)

Fragment B = DNA fragment from Escherichia coli encoding polyphosphate kinase;

Nucleotides 187-2253 (ppk) of the polyphosphate kinase gene (Akiyama et al., J. Bioi. Chem. 1992, 2667 (1992), 22556-22561); orientation relative to the promoter: sense The arrow shows the direction of transcription of the ppk gene.

C = C fragment: nucleotides 11748-11939 of the T-plasmid pTiACH5 Ti plasmid (Gielen et al., EMBO J. 3 (1984), 835-846).

Figure 2 shows the plasmid pACK

The thin line corresponds to plasmid pUC19 and the thick line corresponds to the coding region of the ack gene from Methanosarcina thermophila-biA (Latimer and Ferry, J. Bacteriol., 175 (1993)).

6822-6829). The arrow shows the direction of transcription of the ack gene.

Figure 3 shows plasmid p35S-ACK

Structure of the plasmid:

A = Fragment A: CaMV 35S promoter, nucleotides 6909-7437 (Franck et al., Cell 21 (1980) 285-294).

B = fragment B: DNA fragment from Methanosarcina thermophila encoding acetate kinase; nucleotides 1314-2748 of the acetate kinase gene (ack) (Latimer and Ferry, J. Bacteriol. 175 (1993), 6822-6829);

Orientation towards the promoter: sense The arrow indicates the direction of transcription of the ack gene

C = C fragment: nucleotides 11748-11939 of the Ti-plasmid pTiACHS Ti plasmid (Gielen et al., EMBO J. 3 (1984), 835-846).

Figure 4 shows transformed potato plants relative to untransformed potato plants.

Plants of the potato line JP1 35 transformed with the plasmid p35S-PPK (background) are shown for two untransformed plants (foreground). The plants are about 84, and are kept in a greenhouse.

The following examples illustrate the present invention. The following methods are used in the examples:

1. Cloning procedure

The vector pUC19 for cloning in E. coli

For plant transformation, the gene construct is cloned into the pBinAR binary vector (Hofgen and Willmitzer, Plant Sci. 66 (1990), 221-230).

2. Bacterial strains

For pUC19 vector and pBinAR vector construct, £. coli strain DH5a (Bethesda Research Laboratories, Gaithersburgh, USA).

Transformation of plasmids into potato was accomplished using pGV2260 of the tumor strain C58C1 (Deblaere et al., Nucl. Acids

Gap. 13 (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens

DNA transfer was performed using the direct transformation procedure of Höfgen & Willmitzer (1988, Nucleic Acids 16: 9877). The transformed agrobacterial plasmid DNA was isolated according to the method of Bimbomim & Doly (1979, Nucleic Acids Res. 7, 1513-1523) and analyzed by gel electrophoresis after appropriate restriction.

4. Transformation of potatoes

10 small leaves of potato sterile culture (Solanum tuberosum L.cv. Desirée) were spiked and placed in 10 ml MS medium (Murashige & Skoog, Physiol. Plant 15, 473, 473) containing 2% sucrose, which was selected overnight with Agrobacterium tumefaciens. contains 50 μΐ of its culture

EN 221 515 Β1 glaze. After gentle shaking for 3-5 minutes, the leaves are incubated for another two days in the dark. The leaves were then loaded with 1.6% glucose, 5 mg / L naphthyl acetic acid, 0.2 mg / L benzylaminopurine, 250 mg / L claforant, 50 mg / L kanamycin or 1 mg / L higramycin B and 0.80% Bacto Agar medium for callus induction. After incubation for one week (25 ° C, 3000 lux), the leaves were treated with 1.6% glucose, 1.4 mg / l zeatin ribose, 20 mg / l naphthyl acetic acid, 20 mg / l gibberellic acid, 250 mg / l claforan, 50 mg / 1 kanamycin or 3 mg / l higramycin B and 0.80% Bacto Agar to induce shoot.

5. Radiolabeling of DNA fragments

Radiolabeling of DNA fragments is performed using a DNA random primer kit from Boehringer (Germany) according to the manufacturer's instructions.

6. Northern imprint analysis

RNA was isolated from leaf tissue of plants using a standard procedure. 50 pg RNA was separated on agarose gel (1.5% agarose, 1 χ MÉN buffer, 16.6% formaldehyde). Wash the gel briefly with water after running the gel. RNA was transferred to a Hybond N (Amersham, UK) nylon membrane using 20 x SSC with a capillary imprint. The membrane was then heated in an oven at 80 ° C for 2 hours under vacuum.

The membrane was pre-hybridized in NSEB buffer for 2 hours at 68 ° C and then overnight in NSEB buffer at 68 ° C in the presence of a radiolabeled probe.

7. Plant Maintenance

Light period 16 hours, 2500 lux and 22 ° C dark period 8 hours at 15 ° C humidity 60%

Example 1

Construction of plasmid p35S-PPK and introduction of the plasmid into the genome of potato plants

To construct plasmid p35S-PPK, a DNA fragment encoding polyphosphate kinase from E. coli was first amplified by PCR (Poly20 merase Chain Reaction). For this purpose, genomic DNA from cells of E. coli DHSalpha strain was isolated using standard methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, 1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY). Two oligonucleotides

Oligo 1 5'-AAGGATCCAGGAACCCGGGCCATGGGTCAGGAAAAG-3 '(SEQ ID NO: 1) and

Oligo 2 5'-GGGGATCCCGGGCCATGGGTTATTCAGGTTG-3 ', using a DNA fragment of about 2.1 kb in length, which contains nucleotides 187-2253 of the DNA sequence (ppk gene), encoding the E. coli-béA human polyphosphate kinase and J. Biol. Chem. 267 (1992), 22558) - amplified by PCR. With oligonucleotide number 1, which is partially complementary to the 5 'end of the ppk gene, restriction sites for the BamHI and SmaI restriction endonucleases are introduced at the 5' end of the amplified DNA fragment. By using oligonucleotide number 2, which is partially complementary to the 3 'end of the ppk gene, restriction sites for the BamHI, SmaI and NcoI restriction endonucleases are introduced at the 3' end of the amplified fragment. The DNA fragment obtained from the PCR reaction was digested with BamHI and digested with BamHI-digested pBinAR binary vector (Hofgen and Willmitzer, Plant Sci. 66 (1990), 221-230). The binary vector pBinAR is a derivative of the binary vector pBin19 (Bevan, Nucl. Acids Sl 12 (1984) 8711-8721). The pBinAR vector is constructed as follows:

A 529 bp fragment containing the 6909-7437 nucleotides of the cauliflower mosaic virus 35S promoter (Franck et al., Cell 21 (1980), 285-294) was isolated as an EcoRI / KpnI fragment from plasmid pDH51 (Pietraak et al., Nucl. Acids Slit). 14,5857-5868) and ligated between the EcoRI and KpnI sites of the polynucleotide pBin19. This results in plasmid pBin19.

With the help of PvuII and HindIII restriction endonucleases, a 192 bp long plasmid and a pTiACHS Ti plasmid

A fragment containing the polyadenylation signal of gene 3 of its T-DNA (Gielen et al., EMBO J. 3, 835-846) was isolated from plasmid pAGV40 (Herrera-Estrella et al., Natura 303, 209-213) (11749- 11939 nucleotides). Following addition of SphI binding sites to the Pvul site, the fragment is ligated between the SphI and HindIII sites in pBin19-A. This results in the pBinAR vector

By restriction and sequence analysis, recombinant vectors are identified in which the amplified DNA fragment is inserted into the vector by binding to the 35S promoter in the sense coding region of the polyphosphate kinase from E. coli. The resulting plasmid p35S-PPK is shown in Figure 1.

By inserting the amplified DNA fragment, an expression cassette is constructed which is constructed from fragments A, B and C as follows:

Fragment A (529 bp) contains the 35S promoter of Cauliflower Mosaic Virus (CaMV). The fragment contains nucleotides 6909-7437 of CaMV 55 (Franck et al., Cell 21 (1980) 285-294).

Fragment B contains the coding region for polyphosphate kinase from E. coli and 1872253 nucleotides of the polyphosphate kinase gene (Akiyama et al., 1992, J. Biol. Chem. 267, 22556-22561). This is the encoder

The HU1 211,515 B1 region is ligated to the 35S promoter in sense orientation.

Fragment C (192 bp) contains the polyadenylation signal of the TDNA gene 3 of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The plasmid p35S-PPK has a size of about 13 kb.

Since the coding region of the polyphosphate kinase gene from E. coli used does not contain any signal sequence, the expressed protein must be present in the cytoplasm of the transformed cells. 10

The plasmid was transferred to potato plants by agrobacterial-mediated transformation as described above. Whole plants are regenerated from the transformed cells. In this way, 40 transformed plant lines are generated, of which 7, in particular JP116, JP126, JP129, JP133, JP133, JP134 and JP135, are analyzed in detail.

As a result of the transformation, the transgenic potato plants show expression of the polyphosphate kinase gene from E. coft. Expression is detected by Northem imprint analysis. For this purpose, RNA was isolated from the tissue of transgenic plants, separated by electrophoresis, transferred to a nylon membrane, and transfected with E. coli. cofi is hybridized to the radioactive 25 coding region of the ppk gene. Of the 7 transgenic potato lines mentioned above, only one plant is tested for expression of the ppk gene.

Each plant clearly shows the expression of the ppk gene from E. coli. In contrast, no transcripts are detected in wild-type plants.

Compared to wild type plants, plants are higher per plant! yield (g tuber / plant) as shown in Table 35 below.

n = number of plants used for analysis g / plant = average weight of tuber / plant Transformed plants produced no more, but significantly larger tubers compared to wild type plants.

The starch content of the tubers of the transformed plants corresponds to the starch content of the tubers of the wild-type plants (determined by measuring the density of the tubers).

The transformed plants further show early flowering compared to wild type plants as shown in the tables below.

II. spreadsheet

n % of plants JP1 16 14 33.3 JP1 26 14 46.6 JP1 29 14 28.6 JP1 31 14 35.7 JPl 33 14 57.1 JPl 34 14 35.7 JPl 35 14 64.3 Wild type 14 0.0

III. spreadsheet

Table I

n g / inc. JP1 16 8 113.88 JP1 26 8 115.13 JP1 29 8 111.63 JP1 31 8 132.50 JPl 33 8 122.38 JPl 34 8 122.50 JPl 35 8 117.25 Wild type 8 102.38.

n % of plants JP1 16 14 66.7 JP1 26 14 46.6 JP1 29 14 35.7 JP1 31 14 71.4 JPl 33 14 61.5 JPl 34 14 42.9 JPl 35 14 64.3 Wild type 14 83

£

3t

I

The following table shows the tuber yield in g / plant compared to non-transformed plants (wild type) transformed with p35S-PPK.

Greenhouse plants of strains JP116, JP1 26, JP1 29, JP1 31, JP133, JP134 and JP135 and wild-type plants of the Desirée potato variety were examined. All the potatoes. week after harvesting the plants in the ground.

Tables H and ΠΙ illustrate the early flowering of plants transformed with plasmid p35S-PPK compared to untransformed plants (wild type).

Transformed potato lines JP116, JP1.26, JP1 29, JP1 31, JP1 33, JP1 34 and JP1 35, as well as wild-type Desírée potatoes were grown under greenhouse conditions. Table ΙΙ shows the percentage of test plant lines and wild-type plants that bloom 80 days after the potato was planted in the ground. Table ΙΙ shows the percentage of tested plant lines and wild-type plants that were planted after the potato was planted in the ground

They bloomed 84 days.

n = number of plants tested% plants = number of flowering plants

Transformed plants flower on average 1-2 weeks earlier than wild-type plants, which were maintained under the same conditions.

HU 221 515 Bl

Example 2

Construction of plasmid p35A-ACK and introduction of the plasmid into the potato plant genome

To construct p35S-ACK, a DNA fragment (ack gene) encoding the acetate kinase from Methanosarcina thermophila was isolated. The gene was inserted into the SmaI site of plasmid pUC19. The construction of this pUC plasmid is described in detail by Latimer and Ferry (J. Bacteriol. 175 (1993), 6822-6829, p. 6823, right column). The plasmid used, referred to as pACK in the context of the present invention, is shown in Figure 2.

A fragment of about 1.45 ld) was isolated from this plasmid by ligation with restriction endonucleases Asp718 and BamHI and ligated into the pBinAR binary vector digested with Asp718 and BamHI. The resulting plasmid is called p35S-ACK and is shown in Figure 3.

By inserting the isolated Asp718 / BamHI DNA fragment, an expression cassette containing fragments A, B, and C is created as follows:

Fragment A (529 bp) contains the 35S promoter of Cauliflower Mosaic Virus (CaMV). The fragment contains nucleotides 6909-7437 of CaMV (Franck et al., Cell 21 (1980) 285-294).

Fragment B contains the acetate kinase coding region of Methanosarcina thermophila-biA. The fragment contains nucleotides 1314-2748 of the sequence (acetyl kinase (ack) gene) presented by Latimer and Ferry (J. Bacteriol. 175 (1993), 6822-6829). This coding region is ligated to the 35S promoter in sense orientation.

Fragment C (192 bp) contains the polyadenylation signal of the T-DNA gene 3 of the Ti plasmid pTiACH5 (Gielen et al., 1984, 835-846). Plasmid p35SACK has a size of about 12.5 kb.

As previously described, the plasmid is transformed into potato cells by agrobacteria-mediated transformation. From the transformed cells the whole plant is regenerated.

As a result of the transformation, the transgenic potato shows the expression of Methanosarcina thermophila -> o \ acetate kinase in the cytosol of the cells.

SEQUENCE LIST (1) GENERAL INFORMATION:

(i) INVENTOR:

(A) NAME: Hoechst Schering AgrEvo GmbH (B) STREET: MiraustraBe 54 (C) CITY: Berlin (E) COUNTRY: DE (F) DIRECTORY: 13509 (ii) TITLE OF THE INVENTION: Transgenic plants characterized by improved biomass production (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READING FORM:

(A) THE MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MSDOS (D) SOFTWARE: Patent Release # 1.0, Version # 1.30 (EPA) (vi) PRIORITY DATA:

(A) APPLICATION NUMBER: DE P 4444 460.5 (B) ISSUED DATE: 29-NOV-1994 (2) DETAILS OF SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 36 base pairs (B) TYPE: nuCleotide (C) TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "Oligonucleotide" (iii) HIP YES (xi) DESCRIPTION OF SEQ ID NO: 1: AAGGATCCAG GAACCCGGGC CATGGGTCAG GAAAAG 36 (2) SEQ ID NO: 2

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 31 base pairs (B) TYPE: nucleotide (C) TYPE: single-stranded (D) TOPOLOGY: linear (ii) MOLECULA TYPE: other nucleic acid (A) DESCRIPTION: / desc = "Oligonucleotide" (iii) HIPOT YES (xi) SEQ ID NO: 2: GGGGATCCCG GGCCATGGGT TATTCAGGTTG

Claims (16)

PATENT CLAIMS 1. A transgenic plant cell, characterized in that an easily mobilizable phosphate pool is produced in the cell by delivery and expression of at least one DNA molecule outside the vacuole, which DNA encodes a protein involved in the synthesis of a phosphate-containing molecule that under natural conditions. is not found in higher plants except vacuoles. The transgenic plant cell of claim 1, wherein the phosphate pool is polyphosphate, acetyl phosphate, phosphono pyruvate or fructose-1-phosphate. Transgenic plant cell according to claim 1 or 2, characterized in that the DNA molecule encodes a protein having the enzymatic activity of a polyphosphate kinase, an acetate kinase, a phosphotransacetylase, a phosphoenol pyruvate mutant or a ketohexokinase. Transgenic plant cell according to claim 3, characterized in that the DNA molecule encoding the polyphosphate kinase is derived from E. coli, Klebsiella aerogenes, Neisseria meningitidis or Synechocystia. HU 221 515 BI Transgenic plant cell according to claim 3, characterized in that the DNA molecule encoding the acetate kinase is derived from E. coli, Methanosarcina thermophila, Haemophilus influenza, Bacillus subtilis or Mycoplasma genitalia. 6. The transgenic plant cell of claim 3, wherein the DNA molecule encoding the phosphotransacetylase is derived from Methanosarcina thermophila, E. coli or Mycoplasma genitalia. Transgenic plant cell according to claim 3, characterized in that the DNA molecule encoding the phosphoenol pituva mutase is derived from Tetrahymena pyriformis. The transgenic plant cell of claim 3, wherein the DNA molecule encoding the ketohexokinase is human or rat. 9. Transgenic plant cell according to any one of claims 1 to 3, characterized in that the cell is a photosynthetically active cell. 10. A transgenic plant, wherein the transgenic plant of claims 1-9. A transgenic plant cell according to any one of claims 1 to 6. 11. The transgenic plant of claim 10, wherein said transgenic plant is characterized by enhanced biomass production relative to untransformed plants. Transgenic plant according to claim 10 or 11, characterized in that it has a modified flowering property compared to untransformed plants. 13. A 10-12. Transgenic plant according to any one of claims 1 to 3, characterized in that the plant is a crop. 14. The transgenic plant of claim 13, wherein the plant is potato. 15. A 10-14. Plant propagation material according to any one of claims 1 to 9, characterized in that the plant propagation material according to claims 1-9. The cells according to any one of claims 1 to 6. 16. Use of DNA molecules encoding proteins for the transformation and expression of plant cells, characterized in that the proteins have enzymatic activity leading to the synthesis of polyphosphate, acetyl phosphate, phosphonopyruvate or fructose-1-phosphate, in plant cells. transformation and expression are feasible.