DE19534759A1 - DNA coding for starch-binding protein - Google Patents

Abstract

A novel DNA mol. (I), encoding a protein (A) that occurs both bound to starch grains and in soluble form in plant cells, is selected from the group of: (i) a DNA mol. encoding a 1464 amino acid sequence; (ii) a DNA mol. that has the coding region of a 4856 bp sequence; (iii) DNA that hybridises with (i) or (ii); (iv) DNA that differs from above due to the degeneracy of the genetic code; and (v) fragments, derivs., or allelic variants of the above DNA. All sequences are given in the specification. Also claimed are: (1) a DNA mol. that encodes an antisense RNA mol. that is complementary to transcripts of (I); (2) a DNA mol. encoding an RNA mol. with ribozyme activity that specifically cleaves transcripts of (I); (3) a vector contg. any of the above DNA; (4) prokaryotic cells contg. any of the above DNA, or a vector of (3); (5) transgenic plant cells contg. any of the above DNA in combination with a heterologous promoter that guarantees transcription in plant cells; (6) plants obtainable by regeneration of the cells of (3); (7) plants contg. the cells of (5); (8) starch obtainable from the cells of (5) or the plants of (6) and (7); (9) an RNA mol. obtainable by transcription of any of the mine; (10) (A) encoded by (I); (11) an antibody that specifically recognises (A).

Description

The present invention relates to DNA molecules containing a Encode starch grain-bound protein, as well as methods and recombinant DNA molecules for the production of transgenic Plant cells and plants that have a modified starch with changed viscosity properties and a synthesize modified phosphate content. The invention also affects those resulting from the procedures transgenic plant cells and plants and those from the starch available in transgenic plant cells and plants.

The polysaccharide starch, which is one of the most important Represents storage substances in the plant kingdom, takes place next to the Food use also wide Use as a renewable raw material for manufacturing industrial products. To the application of this raw material To enable in as many application areas as possible necessary, a large variety of materials and an adaptation to the respective requirements of the industry to be processed to reach.

Although starch comes from a chemically uniform The basic building block, the glucose, is made up of starch is not a uniform raw material. It is about rather a complex mixture of different ones Molecular forms that differ in terms of their Degree of branching and the appearance of branches of the Distinguish glucose chains. One differentiates in particular the amylose starch, an essentially unbranched one Polymer of α-1,4 linked glucose molecules, of which Amylopectin starch, which is a mixture of different strongly branched glucose chains, the Branches through the appearance of α-1,6-glycosidic Links come about.  

The molecular structure of the starch, to a large extent due to the degree of branching, the amylose / amylopectin Ratio, the average chain length as well as that Presence of phosphate groups is determined crucial for important functional properties of the Starch or its aqueous solutions. As important Here, for example, functional properties are too name the solubility, the retrograding behavior, the Film formation properties, the viscosity, the Color stability, the gelatinization properties, d. H. Binding and adhesive properties, as well as the cold stability. The starch grain size can also be used for different applications to be of importance.

The adaptation of the starch isolable from plants certain industrial uses are often included With the help of chemical modifications, which are usually time and are expensive. It therefore seems desirable Finding ways to make plants that one Synthesize starch already in its properties meets the requirements of the manufacturing industry.

Conventional ways of producing such plants consist of classic breeding methods and production of mutants. For example, a mutant has been found in maize generated that changed a strength with Viscosity properties synthesized (US patent 5,331,108), and a maize variety (waxy maize) by breeding established whose strength is almost 100% amylopectin exists (Akasuka and Nelson, J. Biol. Chem. 241 (1966), 2280-2285).

Alternatively, plants that have changed their strength with Synthesize properties with the help of genetic engineering Procedures are generated. For example, it has been described in several cases the genetic modification of Potato plants, with the aim of changing the in the Plants synthesized starch (e.g. WO 92/11376; WO 92/14827). Prerequisite for the application of genetic engineering However, the procedure is the availability of DNA sequences,  whose gene products have an influence on the starch synthesis, the Starch modification or starch degradation.

The present invention is therefore based on the object DNA molecules and methods to make it available enable plants to be modified so that they have a Synthesize strengths that differ in terms of their physical and / or chemical properties of starch synthesized naturally in plants differs and therefore for general and / or special Uses is more appropriate.

This task is accomplished by providing the in the Described embodiments solved.

The present invention thus relates to DNA molecules that a protein with the under Seq ID No. 2 specified Encode amino acid sequence. Such proteins are in the Plastids from plant cells both on starch granules bound before, as well as outside of starch grains in free, d. H. soluble form. The enzyme activity of such Proteins lead to an increased when expressed in E. coli Phosphorylation of the synthesized in the cells Glycogen. The molecular weight of these proteins is in the Range of 140-160 kd when using an SDS Gel electrophoresis is determined.

The present invention further relates to DNA molecules which a DNA sequence with the under Seq ID No. 1 specified Sequence of nucleotides, especially those in Seq ID No. 1 indicated coding region.

The invention also relates to DNA molecules that encode a protein found in plant plastids Cells are partially bound to starch granules, and the with the above-mentioned DNA molecules according to the invention hybridize. The term "hybridization" means in  hybridization under this context conventional hybridization conditions, preferably under stringent conditions, such as in Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These DNA molecules that hybridize with the DNA molecules according to the invention, can in principle be from any organism (i.e. Prokaryotes or eukaryotes, especially from bacteria, Fungi, algae, plants or animal organisms), who has such DNA molecules. They preferably come from from monocotyledonous or dicotyledonous plants, in particular from Useful plants, and particularly preferably from starch storing plants.

DNA molecules with the DNA molecules of the invention hybridize, z. B. from genomic or from cDNA Libraries of different organisms are isolated.

The identification and isolation of such DNA molecules from plants or other organisms can under Use of the DNA molecules or parts according to the invention of these DNA molecules or the reverse complement of these Molecules occur, e.g. B. by means of hybridization Standard procedures (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

As a hybridization sample, e.g. B. DNA molecules used be exactly the or essentially the one under Seq ID No. 1 specified DNA sequence or parts of this sequence exhibit. In the DNA samples used as hybridization samples Fragments can also be synthetic DNA fragments act with the help of common DNA synthesis techniques were prepared and their sequence essentially with the of the DNA molecules according to the invention matches. One has Identified and isolated genes associated with the Hybridizing DNA sequences according to the invention is one Determination of the sequence and an analysis of the properties of the proteins encoded by this sequence.  

The present invention further relates to DNA molecules, whose sequences degenerate based on the genetic code are compared to the sequences of the above-mentioned DNA Molecules that encode a protein found in the plastids plant cells partially bound to starch granules is present.

The invention also relates to fragments and derivatives and allelic variants of the DNA described above Molecules encoding the protein described above. Under Fragments are understood to mean parts of the DNA molecules long enough to contain the protein described encode. The term derivative means in this Context that the DNA sequences of these molecules differ the sequences of the DNA molecules described above on one distinguish one or more positions and a high degree have homology to this DNA sequence. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations to the DNA molecules described above can by Deletion, substitution, insertion or recombination have arisen.

Homology also means that functional and / or structural equivalence between the DNA Molecules or the proteins encoded by them. For the DNA molecules that are homologous to those described above DNA molecules are and derivatives of these DNA molecules represent, there are usually variations of these DNA molecules that represent modifications that perform the same biological function. It can happen are both naturally occurring variations, for example, sequences from other organisms, or Mutations, these mutations occurring naturally may have occurred or by targeted mutagenesis  were introduced. Furthermore, there may be variations are synthetically produced sequences.

The allelic variants can be both naturally occurring variants act as well synthetically produced or by recombinant DNA Techniques generated variants.

The of the different variants of the invention Proteins encoded by DNA molecules share certain common features Characteristics on. For this, e.g. B. enzyme activity, Molecular weight, immunological reactivity, conformation etc. belong, as well as physical properties such. B. that Running behavior in gel electrophoresis, chromatographic Behavior, sedimentation coefficient, solubility, spectroscopic properties, stability, pH optimum, Optimum temperature etc.

The DNA molecules according to the invention can in principle consist of any organism that contains the proteins described expressed, preferably from plants, in particular from starch-synthesizing or starch-storing plants. Z are particularly preferred. B. cereals (such Barley, rye, oats, wheat etc.), corn, rice, peas, Cassava, potato, etc.

The invention further relates to vectors, in particular Plasmids, cosmids, viruses, bacteriophages and others in the Genetic engineering common vectors that are described above DNA molecules according to the invention contain.

In a preferred embodiment, the in the Vectors containing DNA molecules linked to DNA Elements that are transcriptional in prokaryotic or ensure eukaryotic cells.

In a further embodiment, the invention relates Host cells, especially prokaryotic or eukaryotic cells that are one described above  contain DNA molecule according to the invention or a vector. These are preferably bacterial cells.

It has now been found that that by the invention DNA molecules encoded an influence on the protein Starch synthesis or modification, and a change the amount of protein in plant cells Changes in the starch metabolism of plants, especially for the synthesis of a starch with modified physical and chemical properties.

By providing the DNA molecules according to the invention it is therefore possible with the help of genetic engineering To produce plants that have a modified starch synthesize themselves in their structure and their physical and chemical properties of wild-type Plants of synthesized starch differ. To do this the DNA molecules according to the invention with DNA elements linked to the transcription in plant cells ensure and introduced into plant cells.

The present invention thus also relates to transgenic Plant cells that have a DNA molecule according to the invention contain, which is linked to a promoter, which ensures transcription in plant cells and which is heterologous to the DNA molecule.

The transgenic plant cells can according to the expert known techniques to regenerate whole plants. The by regeneration of the transgenic invention Plant cells are also available plants Subject of the present invention. Furthermore are Subject of the invention plants that the above described contain transgenic plant cells. With the transgenic In principle, plants can be plants of everyone trade any plant species, d. H. both monocot as well as dicotyledonous plants. It is preferably about Useful plants, in particular starch-storing useful plants, such as B. cereals (rye, barley, oats, wheat etc.), Rice, corn, peas, cassava and potatoes.  

The transgenic plant cells and plants according to the invention synthesize based on the expression or additional Expression of a DNA molecule according to the invention a starch, compared to starch from wild-type plants, d. H. not transformed plants, is modified, especially in With regard to the viscosity of aqueous solutions of this strength and / or the phosphate content. This is usually with the Starch from transgenic plant cells or plants increased, which changes the physical properties of starch will.

The present invention thus also relates to the transgenic plant cells and plants according to the invention available strength.

The invention further relates to the transcription of the RNA molecules obtainable according to the invention and proteins by the DNA molecules of the invention be encoded. These are preferably vegetable, core-encoded proteins found in the plastids are localized. These enzymes are found in the plastids both bound to the starch grains before and free. The corresponding proteins from Solanum tuberosum show in SDS gel electrophoresis has a molecular weight of 140-160 kd and lead to a when expressed in E. coli increased phosphorylation of the synthesized in the cells Glycogen.

The invention also relates to antibodies which specifically recognize a protein according to the invention. It can are both monoclonal and polyclonal Trade antibodies.

It has also been found that it is possible to Properties of the starch synthesized in plant cells by affecting that the amount of proteins that  are encoded by the DNA molecules according to the invention, in the cells is reduced. This reduction can for example by antisense expression of the DNA molecules according to the invention, by expression of suitable Ribozymes or by means of cosuppression.

The present invention thus also relates to DNA Molecules encoding an antisense RNA that are complementary is to transcripts of a DNA molecule according to the invention. Around an antisense in transcription in plant cells Such DNA molecules are at least effective 15 bp long, preferably longer than 100 bp and especially preferably longer than 500 bp, but usually shorter than 5000 bp, preferably shorter than 2500 bp.

In another embodiment, the present relates Invention DNA molecules containing an RNA molecule Encode ribozyme activity that specifically transcripts a cleaves DNA molecule of the invention.

Ribozymes are catalytically active RNA molecules that are in the Are able to target RNA molecules and specific sequences columns. With the help of genetic engineering methods it is possible to change the specificity of ribozymes. It there are different classes of ribozymes. For the practical application with the aim of creating a transcript targeting specific genes is preferred Representative of two different groups of ribozymes used. One group is formed by ribozymes, the are assigned to the type of group I intron ribozymes. The second group is formed by ribozymes, which as characteristic structural feature a so-called Show "hammerhead" motif. The specific detection of the Target RNA molecule can be modified by changing the Sequences that flank this motif. These sequences determine the base pairing with sequences in the target molecule Where the catalytic reaction and thus the The target molecule is cleaved. Since the  Sequence requirements for an efficient split extremely are low, it is in principle possible to use specific ribozymes for practically any RNA molecule. To make DNA molecules that encode a ribozyme that specifically transcripts of a DNA molecule according to the invention cleaves, for example, a DNA sequence that a Coded domain of a ribozyme, on both sides with Linked DNA sequences that are homologous to sequences of the Target enzyme. As sequences covering the catalytic domains encode, for example, the catalytic Domain of the satellite DNA of the SCMo virus (Davies et al., Virology 177: 216-224 (1990) or that of satellite DNA the TobR virus (Steinecke et al., EMBO J. 11 (1992), 1525- 1530; Haseloff and Gerlach, Nature 334 (1988), 585-591). The DNA sequences flanking the catalytic domain preferably from the invention described above DNA molecules.

In another embodiment, the present relates Invention vectors containing the DNA molecules described above included, especially those where the described DNA molecules are linked to regulatory DNA Elements related to transcription in plant cells guarantee.

The present invention further relates to host cells which contain the described DNA molecules or vectors. The Host cell can be a prokaryotic, for example bacterial or eukaryotic cell. Both eukaryotic host cells are preferably plant cells.

The invention further relates to transgenic plant cells which contain a DNA molecule described above, the one encoded antisense RNA or a ribozyme, this DNA The molecule is linked to DNA elements that make up the Ensure transcription in plant cells. This  Transgenic plant cells can be made using common techniques whole plants can be regenerated. The invention relates thus also plants that are available through regeneration from the described transgenic plant cells, as well Plants that have the described transgenic plant cells contain. In turn, the transgenic plants can are plants of any plant species, preferably around crops, especially starch chilling, as stated above.

By expressing the described DNA molecules, the encode antisense RNA or a ribozyme in the transgenic Plant cells experience a decrease in the amount Proteins encoded by DNA molecules according to the invention endogenously present in the cells. This Reduction surprisingly has a drastic one Change in physical and chemical properties the starch synthesized in the plant cells, especially the viscosity properties of aqueous solutions this strength as well as a decrease in Phosphate content of the starch.

The present invention thus also relates to the described transgenic plant cells and plants available strength.

Furthermore, the invention relates to those described by DNA molecules encoded antisense RNA molecules, as well as RNA Molecules with ribozyme activity by transcription are available.

Another object of the present invention is a Process for the production of transgenic plant cells, which in the A modified compared to non-transformed cells Synthesize starch, in which in the plant cells the Amount of proteins is reduced by  DNA molecules according to the invention which are endogenously encoded in in the cells.

In a preferred embodiment, this is done Reduction with the help of an antisense effect. For this DNA molecules according to the invention or parts thereof are in antisense orientation linked to a promoter that the Ensures transcription in plant cells, as well if necessary with a termination signal that the Termination of the transcription and the polyadenylation of the Transcripts guaranteed. To have an efficient antisense To ensure effect in the plant cells should the synthesized antisense RNA has a minimum length of 15 Nucleotides, preferably of at least 100 nucleotides and particularly preferably have more than 500 nucleotides. Furthermore, the DNA sequence encoding the antisense RNA should homologous with respect to the plant species to be transformed be. However, DNA sequences can also be used which have a high degree of homology in cells endogenous have existing DNA sequences, preferably one Homologues of more than 90%, particularly preferably of more than 95%.

In a further preferred embodiment, the Reducing the amount of protein caused by that DNA molecules according to the invention are encoded by a Ribozyme effect. The basic mode of operation of Ribozymes, as well as the construction of DNA molecules that such RNA molecules have already been encoded above described. To use an RNA in transgenic cells To express ribozyme activity are the above described DNA molecules that code for a ribozyme, linked to DNA elements that transcription in ensure plant cells, especially with a Promoter and a termination signal. The in the Ribozymes synthesized by plant cells lead to cleavage  of transcripts of DNA molecules according to the invention which endogenously present in the cells.

The invention also relates to the Plant cells obtainable according to the method of the invention are characterized in that the amount of Proteins is reduced by the invention DNA molecules are encoded, and compared to Wild-type cells synthesize a modified starch.

The invention further relates to plants which are obtainable through regeneration of the plant cells described, and Plants that have the described cells according to the invention contain.

The from the described plant cells and plants available starch is also the subject of the present Invention. Compared to starch from wild-type Plants changed physical and chemical Properties on. For example, this strength in A reduced comparison to the starch from wild-type plants Phosphate content. Furthermore, aqueous solutions show this strength changed viscosity properties.

In a preferred embodiment, the phosphate content the described starch by at least 50%, preferably by at least 75% and particularly preferably by more than 80% in Reduced compared to starch from wild-type plants.

The particular advantage of the strength described lies in the changed viscosity properties of aqueous solutions this strength.

A common test that is used to test the Determining viscosity properties is the so-called Brabender test. This test is carried out under the Use of an apparatus which, for example, as Viscograph E is known. Is manufactured and distributed  this instrument, among others, from Brabender OHG Duisburg (Germany).

The test essentially consists of first heating starch in the presence of water to determine when the starch granules will hydrate and swell. This process, which is also referred to as gelatinization or gelatinization, is based on the dissolution of hydrogen bonds and is accompanied by a measurable increase in the viscosity of the starch suspension. While a further heating after the gelatinization leads to the complete dissolution of the starch particles and a decrease in the viscosity, cooling immediately after the gelatinization typically leads to an increase in viscosity (see FIG. 3). The result of a Brabender test is a curve which shows the viscosity as a function of time, with a temperature increase above the gelatinization temperature and then cooling.

The analysis of a Brabender curve is usually aimed at the determination of the gelatinization temperature, the maximum Viscosity when heated, the viscosity increase at Cooling, as well as the viscosity after cooling. This Parameters are important characteristics that determine the quality one strength as well as its usability for different Determine applications.

The starch, which can be isolated, for example, from potato plants in which the amount of proteins according to the invention in the cells has been reduced by an antisense effect, shows characteristics which differ greatly from the starch which can be isolated from wild-type plants. In comparison to these, it shows only a slight increase in viscosity when heated, a lower maximum viscosity, and a greater increase in viscosity when cooled (see FIGS. 3, 4 and 5).

In a preferred embodiment, the invention thus relates to a starch whose aqueous solutions have the characteristic viscosity properties shown in FIG. 4 or 5. The modified starch, particularly under the conditions mentioned in Example 8a for determining the viscosity with the aid of a Brabender viscometer, has the characteristic that only a slight increase in viscosity takes place during boiling compared to wild-type starch. This offers the possibility of using the starch according to the invention for the production of more concentrated paste.

Furthermore, the modified starch has the property that there is only one after reaching the maximum viscosity slight decrease in viscosity comes. Against it happens cooling to a strong increase in viscosity so that the viscosity is higher than that of a wild-type starch Plants.

The method according to the invention can in principle apply to all Plant species can be applied. Of interest are both monocot and dicot plants, in particular Useful plants and preferably starch-storing Plants such as B. cereal plants (rye, barley, oats, Wheat, etc.), rice, corn, peas, cassava and potatoes.

Under the term "regulatory DNA elements that the Ensure transcription in plant cells " DNA segments within the scope of the present invention understood the initiation or termination of the Enable transcription in plant cells. To the DNA sections representing the initiation of transcription ensure promoters in particular.

For the expression of the various described above DNA molecules according to the invention in plants come in principle each functional promoter in plant cells Consideration. The promoter can be homologous or heterologous on the plant species used. Suitable is  for example the 35S promoter of the Cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812), the one constitutive expression in all tissues of a plant guaranteed and that described in WO / 9401571 Promoter construct. However, promoters can also be used be used only to one by external influences determined point in time (see for example WO / 9307279) or in a certain tissue of the plant Expression of subsequent sequences lead (see e.g. Stockhaus et al., 1989, EMBO J. 8: 2245-2251). Preferentially promoters are used, which in the starch-storing organs of the plants to be transformed are active. In corn, these are the kernels of corn while it is the potatoes are the tubers. To transform the Potato can, in particular, but not exclusively, be the bulb-specific B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can be used.

In addition to promoters, DNA sections can be used to initiate the Transcription also contain DNA sequences that contain another Ensure an increase in transcription, for example so-called enhancer elements.

The term “regulatory DNA elements” can also be used Termination signals include those of correct termination the transcription and addition of a poly A tail serve to the transcript that has a function in the Stabilization of the transcripts is attributed. Such Elements are described in the literature and are arbitrary interchangeable. Examples of such termination sequences are the 3′-untranslated regions that the Polyadenylation signal of the nopaline synthase gene (NOS gene) or the octopine synthase gene (Gielen et al., EMBO J. 8 (1989), 23-29) from Agrobacteria, or the 3'- untranslated regions of the genes of the storage proteins from soybean, as well as that of the genes of the small subunit the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO).  

Furthermore, DNA molecules according to the invention with DNA Sequences are linked in the transcribed area lie and a more efficient translation of the synthesized RNA into the corresponding protein guarantee. Such 5'-untranslated regions can be of viral genes or suitable eukaryotic Genes are obtained or synthetically produced. You can be homologous or heterologous to the used promoter or the DNA sequence to be expressed be.

The introduction of DNA molecules according to the invention into plant Cells are preferably made using plasmids. Plasmids which use a stable integration of the introduced DNA into the plant Ensure genome.

In the examples of the present invention, the binary Vector pBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230) is used. This vector is a Derivative of the binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721), which is commercially available (Clontech Laboratories, Inc., USA).

However, it is also any other plant transformation suitable for vector, in which an expression cassette is inserted and the integration of the expression cassette guaranteed in the plant genome.

To prepare the introduction of foreign genes into higher ones Plants have a large number of cloning vectors available that provide a replication signal for E.coli and a Marker gene for the selection of transformed bacterial cells contain. Examples of such vectors are pBR322, pUC series, M13mp series, pACYc184 etc. The desired one Sequence can be at a suitable restriction site in the vector will be introduced. The plasmid obtained is used for used the transformation of E. coli cells. Transformed E.coli cells are in a suitable Medium cultivated, then harvested and lysed. The  Plasmid is recovered using standard methods. As Analysis method to characterize the plasmid obtained DNA are generally used for restriction analysis and Sequence analysis used. After each manipulation, the Plasmid DNA are cleaved and resulting DNA fragments can be linked to other DNA sequences.

For the introduction of DNA into a plant host cell a variety of techniques are available. This Techniques include transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the Fusion of protoplasts, injection, electroporation of DNA, the introduction of DNA by means of biolistic Method as well as other possibilities.

When injecting and electroporation of DNA into Plant cells in themselves have no special requirements placed on the plasmids used. It can be simple Plasmids such as B. pUC derivatives can be used. But should whole plants from cells transformed in this way to be regenerated is the presence of a selectable Marker gene necessary.

Depending on the method of introducing desired genes into the Plant cells may require additional DNA sequences be. Are z. B. for the transformation of the plant cell if the Ti or Ri plasmid is used, then at least the right boundary, but often the right and left Limitation of the Ti and Ri plasmid T-DNA as a flank region linked to the genes to be introduced.

Agrobacteria are used for the transformation the DNA to be inserted is cloned into special plasmids, either in an intermediate vector or in a binary vector. The intermediate vectors can due to sequences homologous to sequences in the T DNA are, by homologous recombination in the Ti or Ri Plasmid of agrobacteria can be integrated. This contains also the viral necessary for the transfer of T-DNA Region. Intermediate vectors cannot be found in agrobacteria  replicate. Using a helper plasmid, the intermediate vector transferred to Agrobacteriuin tumefaciens become (conjugation). Binary vectors can be used in both Replicate E.coli as well as in agrobacteria. They contain a selection marker gene and a linker or polylinker, which from the right and left T-DNA border region be framed. You can go straight into the agrobacteria can be transformed (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The one for the transformation of agrobacteria plasmids used also contain a Selection marker gene, for example the NPT II gene, which the Selection of transformed bacteria allowed. That as Agrobacterium serving as the host cell is said to be a plasmid which contains a vir region bears included. The vir region is for the Transfer of the T-DNA into the plant cell necessary. Additional T-DNA may be present. That like that transformed agrobacterium is used to transform Plant cells used.

The use of T-DNA for the transformation of Plant cells have been studied intensively and are sufficient in EP 120516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerÿ Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4: 1-46 and An et al., EMBO J. 4 (1985), 277-287. Binary Vectors are already z. T. commercially available, e.g. B. pBIN19 (Clontech Laboratories, Inc., USA).

For the transfer of the DNA into the plant cell Plant explants expediently with Agrobacteriuin tumefaciens or Agrobacterium rhizogenes co-cultivated will. From the infected plant material (e.g. Leaf pieces, stem segments, roots, but also Protoplasts or suspension-cultivated plant cells) can then be in a suitable medium containing antibiotics or contain biocides for selection of transformed cells whole plants can be regenerated again. The so obtained plants can then on the presence of imported DNA are examined.  

Once the inserted DNA is in the genome of the plant cell integrated, so it is usually stable and stays there also in the descendants of the originally transformed Cell received. It usually contains one Selection marker of the transformed plant cells Resistance to a biocide or an antibiotic like Kanamycin, G 418, bleomycin, hygromycin or phosphinotricin u. a. mediated. The individually chosen marker should hence the selection of transformed cells over cells, which lack the introduced DNA.

The transformed cells grow within the plant the usual way (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be dressed normally and with plants that are the same have a transformed genetic makeup or other genetic makeup, to be crossed. The resulting hybrid individuals have the corresponding phenotypic properties.

Two or more generations should be attracted to ensure that the phenotypic trait is stable is retained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other idiosyncrasies have been preserved.

The from the plant cells or plants according to the invention Available starch is suitable due to its properties for various industrial uses.

Basically, starch can be divided into two broad categories divide the starch hydrolysis products and the so-called native strengths. To the hydrolysis products essentially count those via enzymatic or chemical Process received glucose as well as glucose building blocks for other processes, such as fermentation, or even more chemical modifications can be used. In this The range can be the simplicity and cost-effective execution a hydrolysis process, as currently in the essential enzymatic using Amyloglucosidase is important. Leaves underneath  imagine that less use of for Hydrolysis used by changing the enzymes Structure of starch, e.g. B. larger surface of the grain, Easier digestibility due to lower degree of branching or steric, the accessibility for those used Enzyme-limiting structure, at a cost saving can lead.

The uses of the so-called native strengths because of their polymeric structure can be used in divide two major areas:

• (a) Use in the food sector Starch is a classic additive for many Foods where they are essentially the Function of binding aqueous additives takes over or an increase in viscosity or causes increased gel formation. Important Characteristic features are flow and Sorption behavior, the swelling and gelatinization temperature, viscosity and thickening performance, the Solubility of strength, transparency and Paste structure, the heat, shear and acid stability, the tendency to retrogradation, the Ability to film, freeze / thaw stability, the digestibility and the ability to Complex formation with z. B. inorganic or organic Ions.
• (b) Use in the non-food sector The other big area of application is in use the starch as an adjuvant in different Manufacturing processes or as an additive in technical products. The main area of application for the use of starch as an adjuvant the paper and cardboard industry. Strength becomes primarily for retardation (reluctance from Solids), the setting of filler and Fine particles, as a strengthening agent and Drainage used. In addition, the  favorable properties of the starch in relation to the Rigidity, hardness, sound, grip, gloss, the smoothness, the splitting strength and the surfaces exploited.

There are four in the paper making process Areas of application, namely surface, line, mass and spraying to distinguish.

The strength requirements related to the Surface treatments are essentially a high one Whiteness, an adjusted viscosity, a high Viscosity stability, good film formation and a low dust formation. When using the dash is the solids content, an adjusted viscosity high binding power and high pigment affinity important. As an addition to the mass, a quick, even, lossless distribution, high mechanical stability and complete Restraint in the paper flow matters. When using the thickness in the spray area are also adjusted Solids content, high viscosity and a high Attachments important.

A large area of application is, for example, in Adhesives industry where you can find the application in divides four sub-ponds: the use as pure Starch glue, the use of with special Chemical processed starch glues, the use of starch as an additive to synthetic resins and Polymer dispersions and the use of starches as Extender for synthetic adhesives. 90% of Starch-based adhesives are used in the fields Production of corrugated cardboard, production of paper bags, Bags or sachets, manufacture of composite materials for paper and aluminum, production of cardboard and Remoistening glue for envelopes, stamps etc. used.  

Another possible use as an aid and Additive exists in the manufacture of textiles and textile care products. Within the textile industry are the following four uses distinguish: The use of strength as Sizing agents, d. H. as an auxiliary for smoothing and Strengthening the Velcro behavior to protect against the Weaving attacking tensile forces as well as to increase the Abrasion resistance when weaving, as a means of Textile upgrading especially after poor quality other pretreatments such as bleaching, dyeing, etc. as Thickeners in the manufacture of color pastes Prevention of dye diffusion and as an additive for chaining agents for sewing threads.

The starch can also be used as an additive in building materials be used. One example is the production of Gypsum plasterboard, where the mixed in the gypsum paste Starch gelatinized with the water to the surface the plasterboard diffuses and there the cardboard to the Plate binds. Other areas of application are Addition to plaster and mineral fibers. At Ready-mix concrete can be used to delay the strength Binding are used.

Furthermore, the strength for the production of Soil stabilization agents used in artificial Earth movements for the temporary protection of soil particles be used against water. Combination products starch and polymer emulsions are more modern Knowledge in their erosion and incrustation reducing Effect of the previously used products equate, but are significantly less expensive this.

The starch can also be used in crop protection products Change in the specific properties of the preparations be used. For example, strengths become Improve the wetting of crop protection and Fertilizers, for the dosed release of the active ingredients, for  Convert liquid, volatile and / or malodorous Active ingredients in microcrystalline, stable, malleable Substances for mixing incompatible compounds and to extend the duration of action by reducing the Decomposition used.

An important area of application is also in the area the pharmaceutical, medical and cosmetic industries. In the pharmaceutical industry can be considered the strength Binder for tablets or for binder dilution to be used in capsules. The is also suitable Starch as a disintegrant, since after the Swallowing liquid is absorbed and so after a short time swells widely that the active ingredient is released. Medical lubricants and wound powders are another Possible uses. In the field of cosmetics can the strength, for example, as a carrier of Powder additives such as fragrances and salicylic acid be used. A relatively large area of application for the starch is toothpaste.

Also the use of starch as an additive to coal and briquettes is conceivable. Coal can with one Starch additive quantitatively high quality agglomerated or be briquetted, causing early disintegration the briquettes are prevented. The added starch is included Barbecue coal between 4 and 6%, with calorized coal between 0.1 and 0.5%. Furthermore, the Starch as a binder because of its addition to coal and briquette the emission of harmful substances clearly can be reduced.

The strength can also be in the ore and Coal sludge processing used as a flocculant will.

Another area of application is as an addition to Foundry auxiliaries. With different casting processes cores are required that are made from binders Sands. As a binder today  predominantly bentonite used with modified Starches, mostly source starches, is offset.

The purpose of the starch addition is to increase the Flow resistance as well as the improvement of Binding strength. In addition, the source strengths other production requirements, such as in cold water dispersible, rehydratable, good in Sand miscible and high water retention capacity, exhibit.

In the rubber industry, starch can Improvement of the technical and optical quality be used. The reasons are the improvement of the surface gloss, the improvement of the grip and of appearance. For this, strength is given before Cold vulcanization on the sticky rubberized surfaces scattered by rubber materials. It can also be used for Improve the printability of the rubber be used.

Another application of the modified starch exists in the production of leather substitutes.

The following stand out in the plastics sector Areas of application from: the integration of Starch follow-up products in the processing process (starch is only filler, there is no direct binding between synthetic polymer and starch) or alternatively, the integration of starch follow-up products in the manufacture of polymers (starch and polymer go a firm bond).

The use of starch as a pure filler is not compared to the other substances like talc competitive. It looks different if the specific strength properties come into play and hereby the property profile of the end products is significantly changed. An example of this is the Use of starch products in the processing of Thermoplastics, such as polyethylene. Here, the Starch and the synthetic polymer by co-expression  in the ratio of 1: 1 to a "master batch" combined from which with granulated polyethylene under Use of conventional process technologies diverse Products are made. By integrating the Starch in polyethylene films can be increased Permeability to hollow bodies, an improved Water vapor permeability, an improved antistatic behavior, an improved antiblock behavior as well improved printability with aqueous inks can be achieved.

Another option is to apply the starch in Polyurethane foams. With the adaptation of the starch derivatives as well as through process engineering optimization possible the reaction between synthetic polymers and to specifically control the hydroxyl groups of the starch. The result is polyurethane films that are characterized by the Application of starch following property profiles received: a reduction in thermal expansion coefficients, reduction in shrinkage behavior, Improvement in pressure / tension behavior, increase in Water vapor permeability without changing the Water absorption, reduction of flammability and Tear-open density, no dripping of flammable parts, Halogen free and reduced aging. Disadvantages are still present are reduced Compressive strength and reduced impact resistance. It is not only the product development of foils that is possible. Solid plastic products such as pots, plates and Bowls are with a starch content of over 50% to manufacture. Furthermore, the starch / polymer blends the advantage of being a much higher one have biodegradability.

Extraordinary importance continues to be due to their extreme water-binding capacity starch graft poly merisate won. These are products with a backbone of strength and one based on the principle of radical chains mechanism grafted on side rails of a  synthetic monomer. The ones available today Starch plug polymers are notable for better binding and retention capacity of up to 1000 g Water per gram of starch with high viscosity. This Superabsorbers are mainly used in the hygiene sector used, e.g. B. for products such as diapers and pads as well as in the agricultural sector, e.g. B. at Seed pilling.

Crucial for the use of the new genetic engineering changed strength are on the one hand the structure, Water content, protein content, lipid content, fiber content, Ash / phosphate content, amylose / amylopectin ratio, molmas distribution, degree of branching, grain size and shape and Crystallization, on the other hand also the properties that are in the following characteristics flow: flow and sorption behavior, Gelatinization temperature, thickening performance, solubility, Paste structure, transparency, heat, shear and Acid stability, tendency to retrogradation, gel formation, Freeze / thaw stability, complex formation, iodine binding, Film formation, adhesive strength, enzyme stability, digestibility and Reactivity. The Viscosity.

Furthermore, the from the plant cells according to the invention modified starch or plants available chemical modifications are subjected to what further quality improvements for certain of the above described areas of application leads to new ones Areas of application. These chemical modifications are the Basically known to a person skilled in the art. In particular it is doing modifications

• - acid treatment
• - oxidation
• - esterification (formation of phosphate, nitrate, sulfate, Xanthate, acetate and citrate starches. More organic  Acids can also be used for esterification will.)
• - Generation of starch ethers (starch alkyl ether, O- Allyl ether, hydroxyl alkyl ether, O-carboxyl methyl ether, N- containing starch ether, S-containing starch ether)
• - Creation of networked strengths
• - Generation of starch-graft polymers.

Filings

The following plasmids produced and / or used in the context of the present invention were obtained from the German Collection of Microorganisms (DSM) in Braunschweig, Federal Republic of Germany, which is recognized as an international depository, in accordance with the requirements of the Budapest Treaty for the international recognition of the deposit of microorganisms for the purpose of patenting deposited (deposit number; deposit date):
Plasmid pBinAR Hyg (DSM 9505) (10/20/1994)
Plasmid p33-anti-BE (DSM 6146) (08/20/1990)
Plasmid pRL2 (DSM 10225) (09/04/1995)

Media and solutions used

Elution buffer
25mM Tris pH 8.3
250 mM glycine

Dialysis buffer
50 mM Tris-HCl pH 7.0
50 mM NaCl
2mM EDTA
14.7 mM β-mercaptoethanol
0.5 mM PMSF

Protein buffer
50 mM sodium phosphate buffer pH 7.2
10mM EDTA
0.5 mM PMSF
14.7 mM β-mercaptoethanol

Lugol's solution
12 g AI
6 g I₂
ad 1.8 l with ddH₂O

20 × SSC
175.3 g NaCl
88.2 g sodium citrate
ad 1000 ml with ddH₂O
pH 7.0 with 10 N NaOH

10 × MEN
200 mM MOPS
50 mM sodium acetate
10mM EDTA
pH 7.0

NSEB buffer
0.25 M sodium phosphate buffer pH 7.2
7% SDS
1mM EDTA
1% BSA (w / v)

Description of the pictures

Fig. 1 shows the plasmid p35S-anti-RL.

Construction of the plasmid:
A = Fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294)
B = fragment B: approx. 1949 bp Asp718 fragment from pRL1
Orientation to the promoter: anti-sense
The arrow indicates the direction of the open reading frame.
C = fragment C: nt 11748-11939 of the T-DNA of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846)

Figure 2 shows the plasmid pB33-anti-RL

Construction of the plasmid:
A = Fragment A: B33 promoter of the Patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29)
B = fragment B: approx. 1949 bp Asp718 fragment from pRL1
Orientation to the promoter: anti-sense
The arrow indicates the direction of the open reading frame.
C = fragment C: nt 11748-11939 of the T-DNA of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846)

FIG. 3 shows a Brabender curve of an aqueous solution of starch, which was recorded with a Brabender viscograph of the type Viskograph E and was isolated from non-transformed potato plants of the Desiree variety (see Example 8).

Here mean:

Torque Torque
[BE) Brabender units
Temp. Temperature
A Start of gelatinization
B Maximum viscosity
C Start of the hold time
D Start the cooling time
E End of cooling time
F End of the end hold time

The blue line indicates the viscosity; the red the Temperature curve.  

FIG. 4 shows a Brabender curve of an aqueous solution of starch, which was recorded with a Brabender viscograph of the type Viskograph E and was isolated from potato plants which had been transformed with the plasmid p35S-anti-RL (see Example 8). See Fig. 3 for the meaning of the abbreviations.

FIG. 5 shows a Brabender curve of an aqueous solution of starch from potatoes which had been transformed with the plasmid pB33-anti-RL, recorded with a Brabender viscograph of the type Viskograph E (see Example 8). See Fig. 3 for the meaning of the abbreviations.

The examples illustrate the invention.

The following standard techniques were used in the examples applied:

1. Cloning procedure

For cloning in E. coli, the vector pBluescriptSK used.

For the plant transformation the Gene constructions in the binary vector pBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230) and B33- Hyg cloned.

2. Bacterial strains

For the pBluescript vector and for the pBinAR and B33 The E.coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh, USA).

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

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct Transformation using the Höfgen & Willmitzer method (Nucleic Acids Res. 16 (1988), 9877). The plasmid DNA transformed agrobacteria was analyzed using the method of Birnboim & Doly (Nucleic Acids Res. 7 (1979), 1513-1523) isolated and after 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.Desiree) were in 10 ml MS medium (Murashige & Skoog, Physiol. Plans. 15 (1962), 473-497) with 2% sucrose, which 50 µl of a grown under selection Agrobacteriuin tumefaciens overnight culture contained. After Another gentle shaking was carried out for 3-5 minutes Incubation for 2 days in the dark. Then the Callus induction sheets on MS medium with 1.6% Glucose, 5 mg / l naphthylacetic acid, 0.2 mg / l Benzylaminopurine, 250 mg / l claforan, 50 mg / l kanamycin or 1 mg / l hygromycin B, and 0.80% Bacto agar. After a week's incubation at 25 ° C and 3000 lux the leaves for shoot induction on 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 or 3 mg / l hygromycin B, and 0.80% Bacto Agar placed.

5. Radioactive labeling of DNA fragments

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

6. Northern blot analysis

RNA was extracted from leaf tissue according to standard protocols Plants isolated. 50 µg of the RNA were on a Agarose gel separated (1.5% agarose, 1 × MEN buffer, 16.6% formaldehyde). The gel became short after the gel run washed in water. The RNA was analyzed using 20 × SSC Capillary blot on a Hybond N nylon membrane (Amersham, UK) transferred. The membrane was then at 80 ° C under vacuum for two hours baked.

The membrane was placed in NSEB buffer for 2 h at 68 ° C pre-hybridized and then transferred to NSEB buffer Night at 68 ° C in the presence of the radioactively labeled Sample hybridizes.

7. Plant husbandry

Potato plants were kept in the greenhouse under the following conditions:
Light period 16 h at 25000 lux and 22 ° C
Dark period 8 h at 15 ° C
Humidity 60%

example 1 Isolation of starch-bound proteins from Potato starch

Isolation of starch-bound proteins from Potato starch was made by electroelution in one Elution device, which is analogous to the "Model 422 Electro-  Eluter "(BIORAD Laboratories Inc., USA), but had a much larger volume (approx. 200 ml). 25 g of dried starch in elution buffer added (final volume 80 ml). The strength came from Potatoes due to the anti-sense expression of a DNA sequence required for the starch-bound starch synthase I (GBSS I) coded from potato, an almost amylose-free Produce starch. The suspension was in a water bath 70-80 ° C warmed. Then 72.07 g of urea added (final concentration 8 M) and the volume with Make up the elution buffer to 180 ml. The strength came loose with constant stirring and got a paste-like Consistency. The proteins were removed from the solution using the Elution device electroeluted overnight (100 V; 50-60 mA). The eluted proteins were carefully removed from the Apartment taken. Suspended particles were removed by short Centrifugation removed. The supernatant was one 2-3 times Dialyzed against dialysis buffer at 4 ° C for one hour. Subsequently the volume of the protein solution was determined. The proteins were made by adding ammonium sulfate (90% Final concentration). The addition took place under constant stirring at 0 ° C. The precipitated proteins were Pelleted by centrifugation and in protein buffer added.

Example 2 Identification and isolation of cDNA sequences for Encode starch-bound proteins

The proteins isolated according to Example 1 were used Production of rabbit polyclonal antibodies uses the specifically starch-bound proteins detect.

With the help of such antibodies was then followed Standard methods according to a cDNA expression library  Sequences screened for starch-bound Encode proteins.

The expression library was created as follows:
Poly (A⁺) mRNA was isolated from potato tubers of the potato variety "Berolina" using standard methods. Starting from the poly (A⁺) mRNA, cDNA was prepared using the method of Gubler and Hoffmann (Gene 25 (1983), 263-269) using an Xho I-oligo d (t) ₁₈ primer. After EcoR I linker addition, this was cut with Xho I and oriented ligated into a Lambda ZAP II vector (Stratagene) cut with EcoR I and Xho I. Approx. 500,000 plaques of a cDNA library constructed in this way were screened for sequences which were recognized by polyclonal antibodies which are directed against starch-bound proteins.

To analyze the phage plaques, these were based on Transfer nitrocellulose filter, which was previously in for 30-60 min a 10 mM IPTG solution and then open Filter paper were dried. The transfer took place for 3 h at 37 ° C. Then the filter for 30 min Incubated room temperature in block reagent and twice for 5-10 min in TBST buffer. The filters were created with the polyclonal directed against starch-bound proteins Antibodies in a suitable dilution for 1 h Room temperature or shaken at 4 ° C for 16 h. The Identification of plaques that expressed a protein recognized by the polyclonal antibodies was carried out using the "Blotting detection kit for rabbit antibodies RPN 23 "(Amersham UK) according to the Manufacturer.

Phage clones from the cDNA library containing a protein expressed that recognized by the polyclonal antibodies were continued using standard procedures cleaned.

Using the in vivo excision method were positive Phage clones obtained from E. coli clones double-stranded pBluescript plasmid with the respective  cDNA insert included. After checking the size and the Restriction pattern of the inserts became an appropriate one Clone, pRL1, further analyzed.

Example 3 Sequence analysis of the cDNA insertion of the plasmid pRL1

From an E. coli clone obtained in accordance with Example 2 the plasmid pRL1 was isolated and part of the sequence its cDNA insertion by standard methods using the Dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insertion is approx. 2450 bp long. Part of the nucleotide sequence as well as the the amino acid sequence derived therefrom is shown under Seq ID No. 3rd and Seq ID No. 4 specified.

A sequence analysis and a sequence comparison with known ones DNA sequences showed that the under Seq ID No. 3rd sequence shown is new and not significant Has homology to previously known DNA sequences. The Sequence analysis further showed that the cDNA Insertion is only a partial cDNA in which a Part of the coding region at the 5′-end is missing.

Example 4 Identification and isolation of a complete cDNA that for a starch-bound protein from Solanum tuberosum coded

In order to isolate a complete cDNA corresponding to the partial cDNA insertion of the plasmid pRL1, a further cDNA library was produced. This was a lock cell-specific cDNA library from Solanum tuberosum, which was constructed as follows:
First, epidermal fragments of leaves of potato plants of the Desiree variety were essentially processed using the method of Hedrich et al. (Plant Physiol. 89 (1989), 148) by harvesting approximately 60 leaves from six-week-old potato plants kept in the greenhouse. The midrib was removed from the leaves. Then the leaves were crushed in a large "Waring blender" (volume 1 liter) in chilled distilled H₂O four times for 15 seconds each at the highest level. The suspension was filtered through a nylon sieve with a pore size of 220 μm (Nybolt, Zurich, Switzerland) and washed several times with cold distilled water. The suspension was again filtered through a 220 micron nylon sieve and washed extensively with cold distilled water. The residue (epidermal fragments) was placed in a smaller "waring blender" (volume 250 ml) and ground four times in distilled water and ice for 15 seconds each at a low setting. The suspension was filtered through a 220 micron nylon sieve and washed extensively with cold distilled water. The epidermis fragments (residue) were examined microscopically for contamination by mesophyll cells. If this was the case, the shredding step was repeated in the small "Waring blender".

The unlocking of the guard cells of the epidermal fragments was done by crushing in liquid nitrogen in one chilled mortar for about 2 h. To check the Digestion of the locking cells were regularly carried out taken and checked microscopically. After 2 hours or when a sufficiently large number of lock cells unlocked the resulting powder was put into a reaction vessel (Volume 50 ml) transferred and in a volume of GTC buffer (Chirgwin et al., Biochem. 18 (1979), 5294-5299) added. The suspension was centrifuged and the Supernatant by Miracloth (Calbiochem, La Jolla, California) filtered. The filtrate was as in Glisin et al. (1974, Biochemistry 13: 2633-2637) and Mornex et al.  (J. Clin. Inves. 77 (1986), 1952-1961) for 16 hours one Subjected to ultracentrifugation. After centrifugation the RNA precipitate was taken up in 250 ul GTC buffer. The RNA was determined by adding 0.05 volumes of 1 M acetic acid and 0.7 volume of ethanol precipitated. The RNA was centrifuged and the precipitate with 3 M sodium acetate (pH 4.8) and 70% ethanol washed. The RNA became short dried and dissolved in DEPC-treated water.

The isolated RNA was converted into poly A⁺- RNA isolated. Based on the poly (A⁺) mRNA, the Method by Gubler and Hoffmann (Gene 25 (1983), 263-269) using an Xho I oligo d (t) ₁₈ primer cDNA produced. After EcoR I linker addition with Xho I cut and oriented into one with EcoR I and Xho I cut lambda ZAP II vector (Stratagene, GmbH, Heidelberg, Germany). The packaging in Phage heads were made using the Gigapack II Gold kit’s (Stratagene, GmbH, Heidelberg, Germany) after the Manufacturer information.

Such a cDNA library was used to Standard procedures isolated and purified phage clones that hybridize with the cDNA insert from plasmid pRL1. Using the in vivo excision method were positive Phage clones obtained from E. coli clones double-stranded pBluescript plasmid with the respective cDNA insert included. After checking the size and the Restriction patterns of the inserts became suitable clones restriction mapping and sequence analysis subjected. The plasmid pRL2 was made from a suitable clone (DSM 10225) isolated, which contains a complete cDNA, for a starch-bound protein from potato coded.  

Example 5 Sequence analysis of the cDNA insertion of the plasmid pRL2

The nucleotide sequence of the cDNA insert of the plasmid pRL2 was determined as described in Example 3. The insertion is 4856 bp long. The nucleotide sequence and the one from it deduced amino acid sequence is in Seq ID No. 1 or Seq ID No. 2 specified.

Example 6 Construction of the plasmid p35S-anti-RL and introduction of the Plasmids in the genome of potato plants

An approximately 1800 bp DNA fragment was isolated from the plasmid pRL1 using the restriction endonuclease Asp718. This corresponds to that under Seq ID No. 3 DNA sequence shown and contains part of the open reading frame. The fragment was ligated into the binary vector pBinAR cut with Asp718 (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230). This is a derivative of the binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). pBinAR was constructed as follows:
A 529 bp fragment comprising nucleotides 6909-7437 of the 35S promoter of the Cauliflowermosaic virus (Franck et al., Cell 21 (1980), 285-294) was extracted from the plasmid as EcoR I / Kpn I fragment pDH51 (Pietrzak et al., Nucl. Acids Res. 14, 5857-5868) isolated and ligated between the EcOR I and Kpn I sites of the polylinker of pBin19. The plasmid pBin19-A was formed.

From plasmid pAGV40 (Herrera-Estrella et al., Nature 303, 209-213) was carried out using the restriction endonucleases Pvu II and Hind III isolated a 192 bp fragment that the Polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3, 835-846)  (Nucleotides 11749-11939). After adding Sph I linkers the Pvu I interface was the fragment between the Sph I and Hind III sites pBin19-A ligated. Here pBinAR was created.

With the aid of restriction and sequence analyzes, recombinant vectors were identified in which the DNA fragment is inserted into the vector in such a way that part of the coding region of the cDNA insert from pRL1 is linked in anti-sense orientation to the 35S promoter. The resulting plasmid, p35S anti-RL, is shown in Figure 1.

The insertion of the cDNA fragment creates an expression cassette which is constructed from fragments A, B and C as follows:
Fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV). The fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck et al., Cell 21 (1980), 285-294).

In addition to flanking areas, fragment B contains one Part of the protein coding region of the cDNA insert the plasmid pRL1. This was described as Asp718 fragment isolated from pRL1 and in anti-sense Orientation to the 35S promoter fused in pBinAR.

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

The size of the plasmid p35S-anti-RL is approximately 12.8 kb.

The plasmid was mediated using Agrobacteria Transformation in potato plants transferred as above described. The transformed cells became whole Plants regenerate. The transformed plants were cultivated under greenhouse conditions.

Checking the success of the genetic modification of the Plants were done by analyzing the total RNA in one Northern blot analysis for the disappearance of the to cDNA complementary transcripts. For this total RNA was made Leafing transformed plants using standard methods  isolated, gel electrophoresis on an agarose gel separated, transferred to a nylon membrane and with a radioactively labeled sample that hybridizes the under Seq ID No. 1 shown sequence or a part this sequence. In about 5-10% of the transformed Plants lacked the band in the Northern blot analysis the specific transcript of the Seq ID No. 1 represented sequence. These plants became Starch quality analysis used.

Example 7 Construction of the plasmid pB33-anti-RL and introduction of the Plasmids in the genome of potato plants

An approximately 1800 bp long DNA fragment was isolated from the plasmid pRL1 with the aid of the restriction endonuclease Asp718 and comprises part of the open reading frame of the cDNA insert and ligated into the vector B33-Hyg cut with Asp718. This vector was made as follows:
The 35S promoter was removed from the vector pBinAR Hyg (DSM 9505) using the restriction endonucleases EcoR I and Asp718. An approximately 1526 bp long fragment comprising the B33 promoter was isolated from the plasmid p33-anti-BE (DSM 6146) with the aid of EcoR I and Asp718 and was converted into the vector pBinAR Hyg (DSM 9505).

The insertion of the cDNA fragment into the Asp718 site of the plasmid B33-Hyg results in an expression cassette which is constructed from fragments A, B and C as follows ( FIG. 4):
Fragment A contains the B33 promoter from sol on um tuberosum (EP 3775 092; Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).

In addition to flanking areas, fragment B contains one Part of the protein coding region of the cDNA insert the plasmid pRL1. This was described as Asp718 fragment isolated from pRL1 and in anti-sense Orientation to the B33 promoter fused in B33 hyg.

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

The size of the plasmid pB33-anti-RL is approximately 12.8 kb. The plasmid was mediated using Agrobacteria Transformation in potato plants transferred as above described. The transformed cells became whole Plants regenerate. The transformed plants were cultivated under greenhouse conditions.

Checking the success of the genetic modification of the Plants were done by analyzing the total RNA in one Northern blot analysis for the disappearance of the to cDNA complementary transcripts. For this total RNA was made Bulbs of transformed plants using standard methods isolated, gel electrophoresis on an agarose gel separated, transferred to a nylon membrane and with a radioactively labeled sample that hybridizes the under Seq ID No. 1 shown sequence or a part this sequence. In about 5-10% of the transformed Plants lacked the band in the Northern blot analysis Represents transcripts with the cDNA according to the invention hybridize. These plants were turned into tubers Starch isolated and as described in Example 8 analyzed.

Example 8 Analysis of the transformed potato plants

The transformed according to Example 6 and Example 7 Potato plants have been evaluated for the properties of the  synthesized starch examined. The analyzes were on various lines of potato plants carried out the with the plasmid p35S-anti-RL or with the plasmid pB33-anti RL had been transformed and those in the Northern blot Analysis of the gang no longer had the transcripts represents with the DNA sequences of the invention hybridize.

a) Determination of the viscosity of aqueous starch solutions

To determine the viscosity of the aqueous solutions of the starch synthesized in transformed potato plants, starch was isolated from tubers of plants which had been transformed with the plasmid p35S-anti-RL or with the plasmid pB33-anti-RL. In each case 30 g of starch were taken up in 450 ml of H₂O and used for the analysis in a Viscograph E (Brabender OHG Duisburg (Germany)). The device was operated according to the manufacturer's instructions. To determine the viscosity of the aqueous starch solution, the starch suspension was first heated from 50 ° C. to 96 ° C. at a rate of 3 ° C. per minute. The temperature was then kept at 96 ° C. for 30 minutes. The solution was then cooled from 96 ° C to 50 ° C at a rate of 3 ° C per min. The viscosity was determined throughout the duration. Representative results of such measurements are shown in the form of graphs in which the viscosity is shown as a function of time in Fig. 3, Fig. 4 and Fig. 5. Figure 3 shows a typical Brabender curve for starch isolated from wild-type plants of the Desiree potato variety. Figures 4 and 5 show a typical Brabender curve for starch isolated from potato plants transformed with plasmid p35S-anti-RL and pB33-anti-RL, respectively. Various characteristic values can be derived from the curves.

The following result for wild type plants characteristic values:

Table 1

The values give mean values from two different ones Measurements again.

In Table 1 and the following Tables 2 and 3 mean:
A: Start of gelatinization
B: Maximum viscosity
C: Start of the hold time
D: start of cooling time
E: end of cooling time
Q: End of hold time.

For plants with the plasmid p35S-anti-RL had been transformed (line P2) following characteristic values:  

Table 2

For plants with the plasmid pB33-anti-RL had been transformed (line P3) following values:

Table 3

From Figs. 3, 4 and 5, it is clear that the starch from transformed plants differs from that of wild-type plants in particular in that only a very slight viscosity increase occurs during boiling. The maximum viscosity for modified starch from transformed plants when boiled is more than 50% below the value of wild-type starch.

On the other hand, the viscosity increases transformed plants isolated starch after the Cooling down more than with wild-type starch.  

b) Determination of the phosphate content of the starch

The phosphate content of the starch was determined by the Amount of phosphate at the C-6 position of Glucose residue was bound was measured. This was done Starch first split by acid hydrolysis and then the content of glucose-6-phosphate by means of of an enzyme test, as described below.

100 mg starch were in 500 ul 0.7N HCl for 4 h at 100 ° C. incubated. After acid hydrolysis, 10 ul of the Approach in 600 ul imidazole buffer (100 mM imidazole, 5 mM MgCl₂ pH 6.9, 0.4 mM NAD⁺) given. The determination of Amount of glucose-6-phosphate in the batch was made by Reaction with the enzyme glucose-6-phosphate dehydrogenase. For this purpose, 1 U glucose-6-phosphate Dehydrogenase (from Leuconostoc mesenteroides (Boehringer Mannheim)) and the amount of NADH formed determined by measuring the absorption at 340 nm.

The content of glucose-6-phosphate / gram starch is in the following table for non-transformed Potato plants of the Desiree variety as well as for two Lines (P1 (35S-anti-RL; P2 (35S-anti-RL)) transgenic Potato plants with the plasmid p35S-anti-RL had been transformed.

Table 4

The following table shows the glucose-6-phosphate content  per gram of starch in potato plants grown with the Plasmid pB33-anti-RL had been transformed in Compared to starch from non-transformed plants (S. tuberosum c.v. Desiree).

Table 5

Plants 7 , 37 , 45 and 31 represent independent transformants which had been transformed with the plasmid pB33-anti-RL. Plant 37 represents line P3, for which a Brabender curve is shown in FIG. 5.

The values show that the phosphate content of the modified starch from transgenic potato plants at least about 50% compared to starch from wild-type Plants is decreased.

Overall, it is similar to that from transgenic potato plants isolated modified starch of corn starch Wild type plants. In comparison to this, she owns the Advantage that it is tasteless and so for different uses in food area is more suitable.  

Example 9 Expression of the cDNA insert of the plasmid RL2 in E. coli (a) Transformation of bacterial cells

To express the cDNA insert of the plasmid pRL2 cells of the E. coli strain DH5α were initially treated with the Plasmid pACAC transformed. This plasmid contains a DNA fragment that contains the ADP-glucose pyrophosphorylase (AGPase) from E. coli, under the control of the lac Z promoter. The fragment was approximately 1.7 kb in size DraI / HaeII fragment from the vector pEcA-15 (see B. Müller-Röber (1992), dissertation, FU Berlin) isolated been and after smoothing the ends in a HindIII linearized pACACI84 vector has been cloned. The Expression of AGPase is said to increase the Glycogen synthesis in transformed E. coli cells cause. The cells transformed in this way are hereinafter referred to as E. coli K1 cells.

To determine the enzyme activity of the by the cDNA Plasmid pRL2-encoded protein, E. coli K1 Cells transformed with the plasmid pRL2. The transformed E. coli cells, both the plasmid pACAC and the plasmid pRL2 are included in the hereinafter referred to as E. coli K2 cells.

The transfer of the plasmid DNA into the bacterial cells was carried out according to the method of Hanahan (J. Mol. Biol. 166: 557-580 (1983). The transformed E. coli Cells were grown on agar culture dishes using the following Crossed out composition:

YT medium with

1.5% Bacto agar
50 mM sodium phosphate buffer, pH 7.2
1% glucose
10 µg / ml chloramphenicol in E. coli K1 cells or
10 µg / ml chloramphenicol and
10 µg / ml ampicillin in E. coli K2 cells.

Escherichia coli cells of the DH5α strain, which are associated with the Plasmid pRL2 + pACAC (E. coli K2 cells) and as Control only with the plasmid pACAC (E. coli K1 cells) were transformed on agar plates dressed. The glycogen formed in the various Cultures were analyzed for the degree of phosphorylation (an C-6 position of the glucose molecule) examined, as in following is described.

(b) Isolation of bacterial glycogen

For the isolation of bacterial glycogen, the after bacterial turf grown from the transformation of each 6 agar plates (⌀ 135 mm) with 5 ml YT medium / plate washed away. The bacterial suspension was at 4500 xg centrifuged for 5 minutes. The bacterial precipitation was resuspended in 10 ml of YT medium. The information the bacteria were made by adding 2 volumes Digestion medium (0.2 N NaOH; 1% SDS) and incubation for 5 minutes at RT. By adding 3 volumes of EtOH abs., 30 minutes incubation at 4 ° C and then This was centrifuged at 8000 gx for 15 minutes Sediments of glycogen. Then the Wash precipitate with 100 ml 70% EtOH and again through a centrifugation step (10 minutes at 8000 gx) sedimented. The washing process was done 4 times repeated.

(c) Determination of the total glycogen content

The isolated and sedimented glycogen was initially by acid hydrolysis (dissolving the precipitate in 2 ml  0.7 N HCl; Incubation for 4 hours at 100 ° C) in the individual glucose molecules split. Of the Glucose content of the solution was coupled by means of enzymatic reaction of a starch test according to information of the manufacturer (Boehringer Mannheim) on one Photometer (Kontron company) at a wavelength of 340 nm determined.

The reaction buffer contains:

100 mM MOPS, pH 7.5
10 mM MgCl₂
2mM EDTA
0.25 mM NADP
1 mM ATP
1 U / ml glucose-6-phosphate dehydrogenase
2 U / ml hexokinase

The measurement was carried out at 25 ° C. with 10 μl glucose solution.

(d) Determination of the glucose-6-phosphate content

To determine the content of the C-6 position phosphorylated glucose molecules were the same Amounts of glucose, each of the different Bacterial cultures used. By adding the same Volumes of 0.7 N KOH to that by means of acid hydrolysis (see above) split into its glucose molecules Glycogens, the solution was neutralized.

The reaction buffer contains:

100 mM MOPS, pH 7.5
10 mM MgCl₂
2mM EDTA
0.25 mM NADP
2 U / ml glucose-6-phosphate dehydrogenase

The measurement was carried out at 25 ° C with 100-150 µl Glucose solution.  

(e) Detection of a bacterial glycogen phosphorylating Enzyme activity

The results of the determination of the phosphate content of the show glycogen synthesized in the bacterial cells that the glycogen of the E. coli cells with the Plasmids pACAC + pRL2 had been transformed, one up to 290 ± 25% increased phosphorylation at C-6- Position of the glucose compared to that Control approach (E. coli cells transformed with the Plasmid pACYC) (see following table)

The degrees of phosphorylation shown here are the Average of at least 6 measurements based on 6 independent transformations and glycogen isolation.  

Sequence listing

Claims (32)

1. DNA molecule which codes for a protein which is present in plant cells both bound to starch granules and in soluble form, selected from the group consisting of:

• (a) DNA molecules, which are suitable for a protein with the under Seq ID No. Encode 2 given amino acid sequence;
• (b) DNA molecules that encode the coding region of Seq ID No. 1 nucleotide sequence given;
• (c) DNA molecules which hybridize with the DNA molecules mentioned under (a) or (b);
• (d) DNA molecules whose sequence is degenerate due to the genetic code compared to the sequences of the DNA molecules mentioned under (a), (b) or (c); and
• (e) Fragments, derivatives or allelic variants of the DNA molecules mentioned under (a) to (d).

2. Vector containing a DNA molecule according to claim 1. 3. The vector of claim 2, wherein the DNA molecule links is with regulatory DNA elements that the Transcription in eukaryotic or prokaryotic Ensure cells. 4. Prokaryotic cell containing a DNA molecule after Claim 1 or a vector according to Claim 2 or 3. 5. Transgenic plant cell containing a DNA molecule Claim 1 in combination with a heterologous Promoter of transcription in plant cells guaranteed.   6. Plant obtainable by regeneration Plant cell according to claim 5. 7. Plants containing plant cells according to claim 5. 8. Starch obtainable from plant cells according to claim 5 or plants according to claim 6 or 7. 9. RNA molecule obtainable by transcription of a DNA Mo. leküls according to claim 1. 10. Protein produced by a DNA molecule according to claim 1 is encoded. 11. Antibody, specifically a protein according to claim 10 recognizes. 12. DNA molecule encoding an antisense RNA that is complementary to transcripts of a DNA molecule Claim 1. 13. DNA molecule encoding an RNA with ribozyme activity that specifically transcripts of a DNA molecule according to claim 1 splits. 14. Vector containing a DNA molecule according to claim 12 or 13. 15. The vector of claim 14, wherein the DNA molecule is combined with regulatory DNA elements that ensure transcription in plant cells. 16. Host cell containing a DNA molecule according to claim 12 or 13 or a vector according to claim 14 or 15. 17. Transgenic plant cell containing a DNA molecule Claim 12 or 13 in combination with regulatory  DNA elements that translate into plant Ensure cells. 18. Transgenic plant obtainable by regeneration of the Plant cell according to claim 17. 19. Transgenic plant containing plant cells according to An Proverbs 17 20. Starch obtainable from plant cells according to claim 17 or plants according to claim 18 or 19. 21. RNA molecule obtainable by transcription of a DNA Molecule according to claim 12 or 13. 22. Process for the production of transgenic plant cells, which synthesize a modified starch, thereby ge indicates that in the cells the amount of proteins is reduced according to claim 10, the endogenous in the Cell to be synthesized. 23. The method according to claim 22, characterized in that reducing the amount of proteins according to claim 10 is achieved in the cells by an antisense effect. 24. The method according to claim 22, characterized in that reducing the amount of proteins according to claim 10 is achieved in the cells by a ribozyme effect. 25. The method according to claim 22, characterized in that reducing the amount of proteins according to claim 10 achieved in the cells by a cosuppression effect becomes. 26. Plant cell obtainable by a method according to a of claims 22 to 25.   27. Transgenic plant obtainable by regeneration of the Plant cell according to claim 26. 28. Transgenic plant containing plant cells according to An Proverbs 26. 29. Starch obtainable from plant cells according to claim 25 or plants according to claim 27 or 28. 30. Starch according to claim 29, characterized in that it compared to those synthesized in wild-type plants Starch has a reduced phosphate content and aqueous solutions of this strength changed Show viscosity properties. 31. Starch according to claim 29 or 30, characterized in that the phosphate content compared to wild starch type plants is reduced by more than 50%. 32. Starch according to one of claims 29 to 31, characterized ge indicates that it comes from potato.