DE69432796T2 - COMBINATION OF DNA SEQUENCES WHICH ENABLES THE FORMATION OF MODIFIED STARCH IN PLANT CELLS AND PLANTS, METHOD FOR THE PRODUCTION OF THESE PLANTS - Google Patents

Description

The present invention relates to a combination of DNA sequences, those in transgenic plant cells and plants for modification the strength formed in the cells leads. The invention further relates to a method for producing genetically modified Plants related to the physical and chemical properties of the starch formed in comparison to the of course formed strength due to the expression artificial introduced DNA sequences changed are that about this method available Plant cells and plants and those available from these plants modified starch.

Polysaccharides are like starch the essential renewable raw materials from plants.

A crucial factor that the Use of renewable raw materials as industrial raw materials standing in the way is the lack of substances in shape, structure or other physico-chemical properties the requirements correspond exactly to the chemical industry. Two special requirements to one for The technical use of suitable raw material is that it is in high Purity is present and is chemically uniform. The latter is important to ensure a homogeneous reaction during processing to ensure.

Although the starch is a polymer of chemical uniform building blocks, the glucose molecules, it is a complex one Mixture of very different molecular forms that differ in terms of their degree of polymerization and the appearance of branches of glucose chains. The degree of branching determines inter alia the physico-chemical properties of the concerned Strength and therefore their suitability for a big Variety of applications. In particular, a distinction is made between amylose starch essentially unbranched polymer from α-1,4 linked glucose molecules, from the amylopectin starch that in turn a complex mixture of differently branched Represents glucose chains. The ramifications come from the appearance of additional α-1.6 links conditions.

In typical plants for starch production, such as corn or potato, the two forms of starch come in a relationship from about 25 parts of amylose to 75 parts of amylopectin.

To the starch raw material to the different adapt to industrial applications, d. H. its physico-chemical To modify properties, it is necessary inter alia, the degree of branching of strength to be able to influence.

With regard to the suitability of a Basic material such as starch for his Application in the industrial sector therefore seems desirable To provide a process for the production of amylogenic plants, the one Strength synthesize that modified compared to the naturally occurring starch is.

In particular, it is desirable to add starch in this way change, that it has a modified degree of branching, i.e. H. a Decrease or increase the degree of branching, creating a more uniform strength with higher or lower amylose content is formed.

An example of another, for the technical Use of starch useful The property is the content of phosphate groups. Phosphate-containing Strength can in a big Number of areas to be applied, e.g. B. in papermaking, the production of textiles, as adhesives, in the food and medical fields. Starch derivatives containing phosphate are also suitable as emulsifiers to be used. Because the in the majority of amylogenic plants Naturally occurring strength contains only a very small proportion of phosphate groups, whereby a special exception is that of underground organs such. B. Roots or potato tubers formed starch were phosphate groups previously introduced using chemical processes. To the additional Expenses in terms of cost and time involved with such procedures to introduce Starch phosphate groups To avoid connected, it seems desirable to have procedures ready to make it possible to Modify plants so that they form an altered starch that has an increased content has phosphate groups.

Regarding the degree of branching of strength it is already known that for certain plant species, for example maize, by mutagenesis, in which individual genes of the plant are inactivated, varieties are produced can be which only contain amylopectin. For potato has been through chemical Mutagenesis in a haploid line also creates a genotype that does not form amylose (Hovenkamp-Hermelink et al., 1987, Theor. Appl. Genet. 75: 217-221). Haploid lines, or the homozygous diploids developed from them or tetraploid lines, but cannot be used for agriculture. For the agriculturally interesting heterozygous tetraploid lines the mutagenesis technique is not applicable because of the presence inactivation of all copies of four different genetic copies a gene is technically not possible is.

There are also varieties of corn and peas known the amylose strength can produce however, the amylose concentration in these plants is only 60-80%. Farther is the mutagenesis process that underlies the production of these varieties lies on other plants such. B. the potato not applicable.

Visser et al. (19911, Mol. Gen. Genet. 225, 289) have further revealed that potato varieties are largely pure amylopectin starch form, with the help of genetic engineering, in particular by antisense inhibition of the gene for the starch-bound starch, can be generated.

In WO 92/14827 there is a branching enzyme the potato reveals. This enzyme is called the Q enzyme (branching enzyme) denoted by Solanum tuberosum. It is also known that with Help of DNA sequences that contain the information for that described in WO 92/14827 Containing the branching enzyme of the potato, transgenic plants are produced can be where the amylose / amylopectin ratio of starch is changed; although the plants described in WO 92/14827 have no starch form a high degree of amylose.

Synthesis, degradation and modification of strength involve a large Number of enzymes whose interactions have only been partially explained so far.

The synthesis takes place in the potato of strength mainly through the action of starch synthase, the essentially ADP glucose as a substrate for a transfer of a glucose residue on the non-reducing end of a polyglucan uses. Which other enzymes are involved in the synthesis of branched starch are, for the most part unknown.

Vdiederum are on the modification and the degradation of strength several enzymes involved:

The starch phosphorylases, the inorganic Using phosphate as a cosubstrate, α-1,4 bonds build up to four units before an α-1.6 Branch off and work from the non-reducing end. The β-amylase as exoamylase has a high specificity for α-1,4 bonds. The least polymerized substrate is maltotetraose. Cause branches one end of chain degradation, with the last α-1,4 bond before the branch point persists (Whelan, 1961, Nature 190, 954-957). The remaining polyglucans can processed by transglycosylases, which are simultaneously hydrolytic and have synthesizing enzyme activity.

Work on the modification of the strength as transglycosylases, the Q enzyme (branching enzyme) and the T enzyme. The minimum substrate for the reaction catalyzed by the T enzyme is α-1,4 maltose, that to the trisaccharide Panose and glucose is implemented, whereby an α-1.6 bond is formed (Whelan, 1961; Abdullah & Whelan, 1960, J. Biochem. 75, 12P). The Q enzyme catalyzes the same Transglycosylation only on glucans of a chain length of at least 40 units. While the presence of multiple Q enzymes in other species such as maize (Singh & Preiss, 1985, Plant Physiol. 79: 34-40) is known, only that in the case of Solanum tuberosum Branching enzyme described in WO 92/14827 was discovered.

Another transglycosylase, the D-enzyme was first described in 1953 (in Nature 172, 158). Peat et al. (1956, J. Chem. Soc., Part XX: 44-55) describe it as one Transglycosylase, the maltodextrin substrates with two or more Transferring units and doing so only α-1.4 bonds generated. The substrate must consist of at least three units be, regarding the acceptor has a low specificity. Takaha et al. (1993, J. Biol. Chem. 268, 1391-1396) describe the purification of the D-enzyme from potatoes (EC 2.4.1.25) as well as the cloning of a cDNA. The purified enzyme accepted Glucose as a recipient the one to be transferred Chain, however, the donor must consist of at least three glucose units be constructed. The authors do not describe what type of bond arises during transglycosylation and assume that this D-enzyme or disproportionation enzyme probably not so much is involved in the modification rather than in the breakdown of starch. So far still unknown whether other enzymes are involved in the modification of the starch are. The effects of the modification are also unknown of activities of the T or D enzyme on the structure of those formed in the cells Strength these effects have not yet been investigated. Since so far the D enzyme was thought to be involved in starch degradation, would of an inactivation or overexpression of for encoding the D enzyme No effect expected on the basic structure of the starch formed become.

about the enzymatic reactions involved in the introduction of phosphate groups in the strenght nothing is known so far. Neither enzymes have been identified, the for the introduction of the phosphate groups are responsible, there is still a clear one Idea of which substrates act as phosphate group donors.

Accordingly, so far no one amylogenic plants successful through genetic engineering modify specifically so that they produce a strength that is related on their physico-chemical properties, for example the starch content or the degree of branching, is modified so that it is for industrial Processing is more suitable than starch, which of course in Plants occurs.

Furthermore, it is not yet known targeted like an amylogenic plant by genetic engineering can be modified so that in this plant formed strength a higher salary has phosphate groups.

It has now surprisingly been found that it through the introduction a combination of known DNA sequences that a branching enzyme or a potato disproportionation enzyme in plant cells encode, enables will produce plants that synthesize a starch that is used in Compared to natural formed strength is modified, whereby this modified strength differs from the naturally formed Strength especially with regard to the degree of branching and the phosphate content different.

• a) der Kodierregion eines Verzweigungsenzyms oder eines Teils davon, und
• b) der Kodierregion eines Disproportionierungsenzyms oder eines Teils davon

besteht, die jeweils in antisense Orientierung an einen Promotor fusioniert sind, so dass deren Transkription nach Einführung in das pflanzliche Genom transgener Pflanzen zu Transkripten führt, die die Synthese des Verzweigungsenzyms und des Disproportionierungsenzyms in den Zellen durch eine antisense Wirkung hemmen, wodurch in den Zellen eine modifizierte Stärke gebildet wird, die sich in Bezug auf den Verzweigungsgrad und den Phosphatgehalt von der natürlich in den Zellen gebildeten Stärke unterscheidet.The present invention therefore provides a preparation comprising a combination of DNA sequences made up of

• (a) the coding region of a branching enzyme or a part thereof, and
• b) the coding region of a disproportionation enzyme or a part thereof

exists, which are each fused in an antisense orientation to a promoter, so that their transcription after introduction into the plant genome of transgenic plants leads to transcripts which inhibit the synthesis of the branching enzyme and the disproportionation enzyme in the cells by an antisense effect, which in the cells a modified starch is formed which differs in terms of the degree of branching and the phosphate content from the starch naturally formed in the cells.

In particular, the degree of branching the modified starch so changed that there is a reduced or increased branching, so a strength with higher or a low proportion of amylose is synthesized. Alternatively, the two coding regions of the branching enzyme and the disproportionation enzyme each coupled to its own promoter in an antisense orientation become and so independent can be transcribed from each other, or they can be linked to a common promoter be merged.

So far, a combination of DNA sequences that encode a branching enzyme, with DNA sequences, encoding a disproportionation enzyme for genetic modification of plant cells and plants with the aim of being genetically modified Plant a starch to form, in particular, in their physico-chemical properties modified in terms of the degree of branching and the phosphate content is not yet described. The effect of the introduction of the new combination of DNA sequences in a plant genome the physicochemical properties of the starch, especially the degree of branching and the content of phosphate groups, is surprising since there is none Clue for involvement of the D-enzyme in starch synthesis and the introduction of Ramifications, or at the introduction of phosphate groups in the strength formed. According to the findings of Peat et al. (1956) the D enzyme does not have the ability for knotting from α-1.6 Bonds.

The combination of the DNA sequences consists preferably of the coding regions of the branch and Disproportionation enzymes from Solanum tuberosum, the coding region of the branching enzyme on the recombinant plasmid p35SH-anti-BE (DSM 9366) or plasmid p35S-anti-BE (DSM 6144) localized sequence, and the coding region of the disproportionation enzyme which is on the recombinant plasmid p35SH-anti-D (DSM 8479) or plasmid p35Santi-D (DSM 9365) localized sequence. It is possible to either the entire To use coding regions or parts of these sequences, these Parts must be long enough to have an antisense effect in the cells. It is possible to parts to a minimum length of 15 bp to be used, preferably with a length of 100 to 500 bp, or, for one effective antisense inhibition, especially sequences with a Length over 500 bp. Usually DNA molecules shorter than 5000 bp are used preferably sequences that are shorter than 2500 bp.

The recombinant DNA sequences lead to simultaneous or sequential introduction to a plant genome in transgenic plants to form transcripts that promote formation of enzymes of starch metabolism change to that a modified starch is formed in the cells, which in comparison with the course starch formed in the cells inter alia an increased Phosphate content and an altered Has degree of branching, in particular a reduced or one increased Degree of branching, which makes the strength formed a higher or lower proportion contains amylose.

For example, if a combination of DNA sequences with the coding region of the branching enzyme Plasmid p35S-anti-BE (DSM 6144) and the coding region of the disproportionation enzyme on plasmid p35SH-anti-D (DSM 8479), or a combination of DNA sequences with the coding region of the branching enzyme on plasmid p35SH-anti-BE (DMS 9366) and the coding region of the disproportionation enzyme Plasmid p35S-anti-D (DSM 9365) simultaneously or in succession is introduced into the plant genome of transgenic plants, does this for the synthesis of transcripts which are the synthesis of the branching enzyme and inhibit the disproportionation enzyme in the cells so that a modified starch is synthesized in the cells different from the of course starch synthesized in the cells in terms of their degree of branching and their phosphate content.

Furthermore, a combination of DNA sequences with the coding region of the branching enzyme and the coding region of the disproportionation enzyme on a plasmid, namely plasmid p35S-anti-D-anti-BE (DSM 9367) when introduced into a plant genome in transgenic plants to form transcripts that promote formation of the branching enzyme and the disproportionation enzyme in the Inhibit cells so that a modified starch is formed in the cells which is compared to that naturally formed in the cells Strength in terms of their degree of branching and their phosphate content.

• A) ein einstufiges Verfahren ist, dass die folgenden Schritte umfasst:
• a) Herstellung einer Kombination von DNA-Sequenzen aus folgenden Teilsequenzen:
• i) je einem Promotor, der in Pflanzen aktiv ist und die Bildung einer RNA im vorgesehenen Zielgewebe oder den Zielzellen sicherstellt;
• ii) je einer Kodierregion eines Verzweigungsenzyms oder eines Teils davon und eines Disproportionierungsenzyms oder eines Teils davon, derart an den unter i) genannten Promotor fusioniert, dass ein Transkript vom nicht-kodierenden Strang gebildet wird (antisense-Fusion) und
• iii) je einer 3'-nichttranslatierten Sequenz, die in Pflanzenzellen zur Beendigung der Transkription sowie zur Addition von poly-A Resten an das 3'-Ende der RNA führt, derart an die unter ii) genannte Sequenz fusioniert, dass die 3'-nichttranslatierte Sequenz an das 3'-Ende des unter ii) genannten nicht-kodierenden Stranges anschließt;
• b) Transfer und Einbau der Kombinationen von DNA-Sequenzen in ein pflanzliches Genom zur Erzeugung transgener Pflanzenzellen und
• c) Regeneration von intakten, ganzen Pflanzen aus den transformierten Pflanzenzellen, oder
• B) ein einstufiges Verfahren, dass die folgenden Schritte aufweist:
• a) Herstellung einer Kombination von DNA-Sequenzen aus folgenden Teilsequenzen:
• i) einem Promotor, der in Pflanzen aktiv ist und die Bildung einer RNA im vorgesehenen Zielgewebe oder den Zielzellen sicherstellt,
• ii) der Fusion der Kodierregion eines Verzweigungsenzyms oder eines Teils davon und der Kodierregion eines Disproportionierungsenzyms oder eines Teils davon, wobei die beiden Kodierregionen so miteinander fusioniert sind, dass beide Kodierregionen in der selben Richtung (Sense oder Anti-sense) ge- lesen werden, und die an den unter i) genannten Promotor so fusioniert sind, dass sie ein Transkript des nicht-kodierenden Stranges der Fusion (Antisense-Fusion) bilden, und
• iii) eine 3'-nichttranslatierte Sequenz, die in Pflanzenzellen zur Beendigung der Transkription sowie zur Addition von poly-A Resten an das 3'-Ende der RNA führt, derart an die unter ii) genannte Sequenz fusioniert, dass die 3'-nichttranslatierte Sequenz and das 3'-Ende des unter ii) genannten nicht-kodierenden Stranges anschließt,
• b) Transfer und Einbau der Kombinationen von DNA-Sequenzen in ein pflanzliches Genom zur Erzeugung transgener Pflanzenzellen, und
• c) Regeneration von intakten, ganzen Pflanzen aus den transformierten Pflanzenzellen, oder
• C) ein zweistufiges Verfahren, bei dem zunächst eine DNA-Sequenz, bestehend aus den folgenden Teilstücken:
• iv) einem Promotor, der in Pflanzen aktiv ist und die Bildung einer RNA im vorgesehenen Zielgeweben oder den Zielzellen sicherstellt,
• v) einer Kodierregion eines Verzweigungsenzyms oder eines Teils davon, das derart an den unter iv) genannten Promotor fusioniert ist, dass ein Transkript vom nicht-kodierenden Strang gebildet wird (antisense-Fusion), und
• vi) einer 3'-nichttranslatierten Sequenz, die in Pflanzenzellen zur Beendigung der Transkription sowie zur Addition von poly-A Resten an das 3'-Ende der RNA führt, derart an die unter v) genännte Sequenz fusioniert, dass die 3'-nichttranslatierte Sequenz an das 3'-Ende des unter v) genannten nichtkodierenden Stranges anschließt, und die in das Genom einer Pflanzenzelle transferiert und eingebaut wird, und dass aus den auf diesem Weg genetisch modifizierten Pflanzen eine ganze Pflanze regeneriert wird, und anschließend durch wiederholte Transformation in Zellen dieser genetisch modifizierten Pflanze eine andere DNA-Sequenz, die aus den folgenden Teilsequenzen besteht:
• vii) einem Promotor, der in Pflanzen aktiv ist und die Bildung einer RNA im vorgesehenen Zielgewebe oder den Zielzellen sicherstellt,
• viii) einer Kodierregion eines Disproportionierungsenzyms oder eines Teils davon, das derart an den unter vii) genannten Promotor fusioniert ist, dass ein Transkript vom nicht-kodierenden Strang gebildet wird (antisense-Fusion), und
• ix) einer 3'-nichttranslatierten Sequenz, die in Pflanzenzellen zur Beendigung der Transkription sowie zur Addition von poly-A Resten an das 3'-Ende der RNA führt, derart an die unter viii) genannte Sequenz fusioniert, dass die 3'nichttranslatierte Sequenz an das 3'-Ende des unter viii) genannten nichtkodierenden Stranges anschließt, und die in das Genom einer Pflanzenzelle transferiert und eingebaut wird, und dass schließlich aus den auf diesem Weg genetisch modifizierten Pflanzenzellen, die beide Kodierregionen oder Teile davon enthalten, eine ganze Pflanze regeneriert wird, oder
• D) ein wie unter C) beschriebenes zweistufiges Verfahren, wobei die unter v) genannte Kodierregion nicht für ein Verzweigungsenzym, sondern für ein Disproportionierungsenzym kodiert, und die unter viii) genannte Kodierregion nicht für ein Disproportionierungsenzym, sondern für ein Verzweigungsenzym kodiert.

The present invention also provides methods for the production of transgenic plant cells and transgenic plants that are capable of synthesizing a modified starch that is different from the starch naturally synthesized in the cells, particularly in terms of their degree of branching and their phosphate content. The transgenic plants can be made by first one of the DNA combinations according to the invention are stably integrated into the genome of a plant cell and then whole plants are regenerated from these transformed plant cells, characterized in that this method:

• A) is a one step process that includes the following steps:
• a) Production of a combination of DNA sequences from the following partial sequences:
• i) one promoter each, which is active in plants and ensures the formation of an RNA in the intended target tissue or cells;
• ii) a coding region of a branching enzyme or a part thereof and a disproportionation enzyme or a part thereof, fused to the promoter mentioned under i) such that a transcript is formed from the non-coding strand (antisense fusion) and
• iii) a 3'-untranslated sequence which leads to the termination of transcription and addition of poly-A residues to the 3 'end of the RNA in plant cells, fused to the sequence mentioned under ii) in such a way that the 3'- untranslated sequence at the 3 'end of the non-coding strand mentioned under ii);
• b) transfer and incorporation of the combinations of DNA sequences into a plant genome to generate transgenic plant cells and
• c) regeneration of intact, whole plants from the transformed plant cells, or
• B) a one-step process that comprises the following steps:
• a) Production of a combination of DNA sequences from the following partial sequences:
• i) a promoter that is active in plants and ensures the formation of an RNA in the intended target tissue or cells,
• ii) the fusion of the coding region of a branching enzyme or a part thereof and the coding region of a disproportionation enzyme or a part thereof, the two coding regions being fused to one another in such a way that both coding regions are read in the same direction (sense or anti-sense), and which are fused to the promoter mentioned under i) such that they form a transcript of the non-coding strand of the fusion (antisense fusion), and
• iii) a 3'-untranslated sequence which leads to termination of transcription and addition of poly-A residues to the 3'-end of the RNA in plant cells, fused to the sequence mentioned under ii) in such a way that the 3'-untranslated Sequence connects to the 3 'end of the non-coding strand mentioned under ii),
• b) transfer and incorporation of the combinations of DNA sequences into a plant genome to produce transgenic plant cells, and
• c) regeneration of intact, whole plants from the transformed plant cells, or
• C) a two-stage process, in which first a DNA sequence consisting of the following sections:
• iv) a promoter which is active in plants and ensures the formation of an RNA in the intended target tissue or cells,
• v) a coding region of a branching enzyme or a part thereof which is fused to the promoter mentioned under iv) in such a way that a transcript is formed from the non-coding strand (antisense fusion), and
• vi) a 3'-untranslated sequence which leads to termination of transcription and addition of poly-A residues to the 3'-end of the RNA in plant cells, fused to the sequence mentioned under v) such that the 3'-untranslated Sequence at the 3 'end of the non-coding strand mentioned under v), and which is transferred and incorporated into the genome of a plant cell, and that a whole plant is regenerated from the genetically modified plants in this way, and then by repeated transformation into Cells of this genetically modified plant have a different DNA sequence, which consists of the following partial sequences:
• vii) a promoter that is active in plants and ensures the formation of an RNA in the intended target tissue or cells,
• viii) a coding region of a disproportionation enzyme or a part thereof which is fused to the promoter mentioned under vii) in such a way that a transcript is formed from the non-coding strand (antisense fusion), and
• ix) a 3'-untranslated sequence which leads in plant cells to the end of transcription and to the addition of poly-A residues to the 3 'end of the RNA, fused to the sequence mentioned under viii) in such a way that the 3' untranslated sequence connects to the 3 'end of the non-coding strand mentioned under viii), and which is transferred and incorporated into the genome of a plant cell, and finally, from the plant cells genetically modified in this way, which contain both coding regions or parts thereof, an entire plant is regenerated, or
• D) a two-stage process as described under C), the coding region mentioned under v) not coding for a branching enzyme but for a disproportionation enzyme, and the coding region mentioned under viii) not for a disproportionation enzyme but for a branching enzyme.

The combination in use coming and mentioned under process steps i), iv) and vii) Promoters can in principle all promoters that are active in plants. For example it possible the 35S promoter of the cauliflower mosaic virus to use the principle of a constitutive expression of downstream DNA sequences in all tissues of transformed plants. preferentially are promoters used in the starch-storing organs of plants to be transformed are active. These are the case with maize Corn kernels, while it is the tubers of potatoes. For the transformation of potatoes can in particular, but not exclusively, the bulb-specific B33 promoter from Solanum tuberosum or another promoter of one Class I patatens are used. The coding regions of branching enzyme and disproportionation enzyme are fused to the promoter such that the 3 'end of the Coding region at the 3 'end of the promoter added becomes. This arrangement is called the antisense orientation. The antisense orientation causes the expression of the Transgenic the non-codogenic strand for the synthesis of a transcript is read. The non-coding transcript can be in a genetic changed Plant neutralize the endogenous coding transcript so that there is no translation into a peptide. This eliminates the enzymatic activity of the branching and disproportionation enzyme in the genetically changed Plants. The success of translation inhibition depends internally alia from the amount of antisense transcripts effective in the cell from. To stabilize the transcripts introduced by the combination of DNA sequences are therefore formed at the coding regions of branching enzyme and disproportionation enzyme a termination and polyadenylation signal attached. This can be the termination signal of the nopaline synthase gene, for example from Agrobacterium tumefaciens.

By the merger of promoter, Coding region of the branching or disproportionation enzyme and termination signal constructs are preferred introduced into plant cells using suitable plasmids. The recombinant plasmids can the combination as a fusion of both coding DNA sequences or Parts of these sequences that are long enough to have an antisense effect to cause contain, or they can each be an element of Combination included. If the recombinant plasmids are both of the DNA sequences referred to above can contain either from a common promoter (process variant B)) or each from its own promoter (process variant A)) be transcribed. If the recombinant plasmids contain both elements the combination can contain transgenic plants, the inventive combination of DNA sequences included, are produced in a one-step process. In the one-stage according to the invention Process (process variant B)) is preferably the plasmid p35S-anti-D-anti-BE (DSM 9367) used. If the recombinant plasmids are each one Containing element of the combination, they can be successively used for transformation are used, so that transgenic plants, the combination of the invention of DNA sequences contained, produced in a two-stage process can be.

In the two-stage according to the invention Processes (process variants C) and D)) are preferably the Plasmids p35S-anti-BE (DSM 6144) and p35SH-anti-D (DSM 8479), or the plasmids p35Santi-D (DSM 9365) and p35SH-anti-BE (DSM 9366) or derivatives thereof. The plasmids are another subject the invention.

The invention also provides plant cells and plants prepared using the method according to the invention can, in their genome one or more of the DNA combinations according to the invention is (are) integrated, and are capable of a modified Strength to synthesize inter themselves from the naturally formed strength alia differs in terms of the degree of branching and the phosphate content.

In principle, the method according to the invention can be applied to all plants. Are of particular interest the plants, the strength form as storage substance, especially agricultural plants. The method according to the invention are preferably used in plants in which a branching enzyme and a disproportionation enzyme involved in the modification of the starch are. It is preferably a potato plant.

The partial sequences from the new combination of DNA sequences which code for the branching enzyme and the disproportionation enzyme of Solanum tuberosum can be replicated by cloning in plasmid vectors of bacteria. Examples of vectors are pBR322, those of the pUC series, the m13mp series etc. The DNA sequences can be provided with linkers which enable simple recloning into other plasmids. For introduction into plants (see example 6) it is preferably, but not exclusively, possible to use binary plasmids which contain a replication signal, e.g. B. for Escherichia coli and for Agrobacterium tumefaciens. If the binary plasmids contain T-DNA sequences, it is particularly easy to transfer the combination of DNA sequences into the genome of dicotyledonous plants. However, other methods are also possible, for example the transformation using ballistic methods which are used for the transformation of monocotyledons (Potrykus, 1991, Ann.

Rev. Plant Mol. Biol. Plant Physiol. 42, 205-225) is used.

As a result of the transfer of the new combination of DNA sequences, each with a promoter, a coding region of the Branching and disproportionation enzyme or a fusion of the two coding regions in antisense orientation to the promoter, and contain a termination / polyadenylation signal it in the transgenic plants to form RNA molecules that by interacting with the endogenous mRNAs of the branching enzyme and the disproportionation enzyme suppress their synthesis. Thereby becomes a strength accessible, those in terms of their degree of branching and their phosphate content is modified.

The one formed in the transgenic plants Strength can with common Methods isolated from plants or from plant cells and after cleaning to produce food and industrial products are processed:

deposits

The plasmid p35-anti-BE (DSM 6144) and the plasmid p35SH-anti-D (DSM 8479) on August 20, 1990 and on August 26, 1993 at the German Collection of Microorganisms (DSM) in Braunschweig, Federal Republic of Germany, according to the regulations of the Budapest Treaty.

The following additional plasmids were deposited on August 10, 1994 at the German Collection of Microorganisms (DSM) in Braunschweig, Federal Republic of Germany, in accordance with the provisions of the Budapest Treaty:

Description of the pictures

• A = Fragment A (529 bp) beinhaltet den 35S Promotor des Blumenkohl-Mosaik-Virus (CaMV), d. h. Nukleotide 6909 bis 7437 des CaMV
• B = Fragment B (2909 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Verzweigungsenzyms von Solanum tuberosum
• C = Fragment C (192 bp) beinhaltet das Polyadenylierungssignal des Gens 3 der T-DNA des Ti-Plasmids pTi ACH5, Nukleotide 11749–11939.

1 shows the 13.6 kb plasmid p35S-anti-BE (DSM 6144). The plasmid contains the following fragments:

• A = fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV), ie nucleotides 6909 to 7437 of the CaMV
• B = fragment B (2909 bp) contains a DNA fragment with the coding region of the branching enzyme of Solanum tuberosum
• C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi ACH5, nucleotides 11749-11939.

• A = Fragment A (529 bp) beinhaltet den 35S-Promotor des Blumenkohl-Mosaik-Virus (CaMV), d. h. Nukleotide 6909 bis 7437 des CaMV
• B = Fragment B (1474 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Disproportionierungsenzyms von Solanum tuberosum
• C = Fragment C (192 bp) beinhaltet das Polyadenylierungssignal des Gens 3 der T-DNA des Ti-Plasmids pTiACH5, Nukleotide 11749–11939.

2 shows the 12.165 kb plasmid p35SH-anti-D (DSM 8479). The plasmid contains the following fragments:

• A = fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV), ie nucleotides 6909 to 7437 of the CaMV
• B = fragment B (1474 bp) contains a DNA fragment with the coding region of the disproportionation enzyme of Solanum tuberosum
• C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5, nucleotides 11749-11939.

• A = Fragment A (529 bp) beinhaltet den 35S-Promotor des Blumenkohl-Mosaik-Virus (CaMV), d. h. Nukleotide 6909 bis 7437 des CaMV
• B = Fragment B (3068 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Verzweigungsenzyms von Solanum tuberosum
• C = Fragment C (192 bp) beinhaltet das Polyadenylierungssignal des Gens 3 der T-DNA des Ti-Plasmids pTiACH5, Nukleotide 11749–11939.

3 shows the plasmid p35SH-anti-BE (DSM 9366). The plasmid is 13.75 kb long and contains the following DNA fragments:

• A = fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV), ie nucleotides 6909 to 7437 of the CaMV
• B = fragment B (3068 bp) contains a DNA fragment with the coding region of the branching enzyme of Solanum tuberosum
• C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5, nucleotides 11749-11939.

• A = Fragment A (529 bp) beinhaltet den 35S-Promotor des Blumenkohl-Mosaik-Virus (CaMV), d. h. Nukleotide 6909 bis 7437 des CaMV
• B = Fragment B (1474 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Disproportionierungsenzyms von Solanum tuberosum
• C = Fragment C (192 bp) beinhaltet das Polyadenylierungssignal des Gens 3 der T-DNA des Ti-Plasmids pTiACH5, Nukleotide 11749–11939.

4 shows the plasmid p35S-anti-D (DSM 9365). The plasmid is 12.2 kb long and contains the following DNA fragments:

• A = fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV), ie nucleus otide 6909 to 7437 of the CaMV
• B = fragment B (1474 bp) contains a DNA fragment with the coding region of the disproportionation enzyme of Solanum tuberosum
• C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5, nucleotides 11749-11939.

• A = Fragment A (529 bp) beinhaltet den 35S-Promotor des Blumenkohl-Mosaik-Virus (CaMV), d. h. Nukleotide 6909 bis 7437 des CaMV
• B = Fragment B (1474 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Disproportionierungsenzyms von Solanum tuberosum
• C = Fragment C (192 bp) beinhaltet das Polyadenylierungssignal des Gens 3 der T-DNA des Ti-Plasmids pTiACH5, Nukleotide 11749–11939
• D = Fragment D (3068 bp) beinhaltet ein DNA-Fragment mit der Kodierregion des Verzweigungsenzyms von Solanum tuberosum.

5 shows the plasmid p35S-anti-D-anti-BE (DSM 9367). The plasmid is 15.23 kb long and contains the following DNA fragments:

• A = fragment A (529 bp) contains the 35S promoter of the cauliflower mosaic virus (CaMV), ie nucleotides 6909 to 7437 of the CaMV
• B = fragment B (1474 bp) contains a DNA fragment with the coding region of the disproportionation enzyme of Solanum tuberosum
• C = fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5, nucleotides 11749-11939
• D = fragment D (3068 bp) contains a DNA fragment with the coding region of the branching enzyme of Solanum tuberosum.

6 shows a "Northern Blot" analysis of the total RNA from potato plants which were produced by a two-stage process, plant cells first using plasmid p35S-anti-BE (DSM 9366) and, after selection and regeneration of the transformants, plant cells of these transformants using plasmid p35SH -anti-D (DSM 9365) were transformed.

The total RNA was examined for the presence of transcripts coding for the branching enzyme or the disproportionation enzyme. The cDNA molecules coding for these enzymes were used as hybridization samples. Volumes 1 and 5: Wild-type potato plants (Solanum tuberosum cv Desirée) Bands 2, 3 and 4: Three different transformed clones produced by the method described above, each containing a DNA combination according to the invention.

The top of the two hybridization signals corresponds to transcripts for encode the branching enzyme, and the lower hybridization signal corresponds to transcripts for encode the disproportionation enzyme.

7 shows a "Northern Blot" analysis of the total RNA from potato plants which were produced using a two-stage process, plant cells first using plasmid p35S anti-D (DSM 9365) and, after selection and regeneration of the transformants, plant cells of these transformants using plasmid p35SH- anti-BE (DSM 9366) were transformed.

The total RNA was examined for the presence of transcripts coding for the branching enzyme or the disproportionation enzyme. The cDNA molecules coding for these enzymes were used as hybridization samples. Volumes 1 and 10: Wild-type potato plants (Solanum tuberosum cv Desirée) Bands 2 to 9: Eight different transformed clones produced by the method described above, each of which contains a DNA combination according to the invention.

The top of the two hybridization signals corresponds to transcripts for encode the branching enzyme, and the lower hybridization signal corresponds to transcripts for encode the disproportionation enzyme.

8th shows a "Northern Blot" analysis of the total RNA from potato plants, which were produced in a one-step process, wherein plant cells are transformed with plasmid p35S-anti-D-anti-BE (DSM 9367) and whole plants are regenerated from the transformed cells were.

The total RNA was examined for the presence of transcripts coding for the branching enzyme or the disproportionation enzyme. The cDNA molecules coding for these enzymes were used as hybridization samples. Volumes 1 and 8: Wild-type potato plants (Solanum tuberosum cv Desirée) Bands 2 to 7: Six different transformed clones produced by the method described above, each of which contains a DNA combination according to the invention.

The top of the two hybridization signals corresponds to transcripts for encode the branching enzyme, and the lower hybridization signal corresponds to transcripts for encode the disproportionation enzyme.

To better understand the examples according to the invention be first the main procedures explained.

1. Cloning procedure

The vector pUC18 was used for cloning (Yanisch-Perron et al. (1985) Gene 33, 103-119).

For the plant transformations were the gene constructs in the binary vector BIN19 (Bevan (1984) Nucl. Acids Res. 12, 8711-8720).

2. Bacterial strains

For the pUC vectors were the E. coli strains BMH71-18 (Messing et al. (1977) Proc. Natl. Acad. Sci. USA 24, 6342-6346) or TB1. TB1 is a recombination-negative, tetracycline-resistant derivative strain JM 101 (Yanisch-Perron et al. (1985) Gene 33, 103-199). The Genotype of the TB1 strain is (Bart Barrel, personal communication): F, traD36, proAB, lacl, lacZdM15, d (lac, pro), SupE, thiS, recA, Srl :: Tn10 (Tcr).

The plant transformation was using the Agrobacterium tumefaciens strain LBA4404 (Bevan (1984) Nucl. Acids Res. 12, 8711-8721), BIN19 derivative.

3. Transformation from Agrobacterium tumefaciens

In the case of BIN19 derivatives, the DNA into the agrobacteria by direct transformation according to the method by Holsters et al. (1978, Mol. Gen. Genet. 163, 181-187). The Plasmid DNA transformed Agrobacteria was analyzed according to the method by Birnboim et al. (1979, Nucl. Acids Res. 7, 1513-1523) and separated by gel electrophoresis after suitable restriction cleavage.

4. Plant transformation

10 small ones wounded with a scalpel leaves a potato sterile culture was placed in 10 ml MS medium with 2% sucrose placed, which 30-50 ul one under Selection contained grown Agrobacterium tumefaciens post-culture. To 3-5 minutes easy shake the Petri dishes were at 25 ° C incubated in the dark. After 2 days the leaves were on MS medium with 1.6% glucose, 2 mg / l zeatinribose, 0.02 mg / l naphthylacetic acid, 0.02 mg / l giberellic acid, 500 mg / l Claforan, 50 mg / l kanamycin and 0.8% Bacto agar. After a week Incubation at 25 ° C and 3000 lux, the Claforan concentration in the medium was reduced by half. The further cultivation was carried out as described by Rocha-Sosa et al. (1989, EMBO Journal 8, 29).

5. Analysis genomic DNA from transgenic plants

Isolation of plant genomic DNA was done according to Rogers et al. (1985, Plant Mol. Biol. 5, 69-76). For DNA analysis 10-20 μg of DNA were found suitable restriction cleavage with the help of "Southern blots" for integration of the DNA sequences to be examined analyzed.

6. Analysis of total RNA from transgenic plants

Isolation of total plant RNA was done according to Logemann et al. (1987, Analytical Biochem. 163, 16-20). For analysis were each 50 μg Total RNA using "Northern Blots "on the presence of the searched for transcripts.

7. Determination of the phosphate content of isolated from transgenic potato plants Strength

In order to determine the phosphate content of starch formed in transgenic plants, starch was isolated from potato tubers and 250 mg of this starch were suspended in 1 ml of 0.7 N HCl. The suspension was incubated at 100 ° C for 4 h. 800 ml of buffer (100 mM MOPS-KOH with a pH of 7.5; 10 mM MgCl ₂ ; 2 mM EDTA) were added to a 100 ml aliquot, the mixture was transferred to a cuvette and 100 ml of 0 were added , 7 N KOH neutralized. The following were then added in succession: NAD (final concentration: 0.4 mM) and glucose-6-phosphate dehydrogenase from Leuconostoc (Sigma) (final volume of the test batch: 1 ml). The change in absorption was measured at a wavelength of 340 nm.

The following examples explain the Subject of the invention without restricting it, initially the Construction of binary Plasmids and then the production of transgenic potato plants that combine the DNA sequence included.

In an analogous procedure, too other crops in which a branching and a disproportionation enzyme on the modification of the strength are involved.

example 1

Construction of the binary plasmid p35S-anti-BE

Corresponding to that in WO 92/14827 the plasmid p35S-anti-BE (DSM 6144) produced, the following steps in particular being carried out: From one in the expression vector λgt11 cDNA bank of tuber tissue from Solanum tuberosum var. Desirée Different clones were identified, one against the other Branching enzyme directed antiserum were recognized (to carry out the immunological determination s. Example 3, "Western Blot"). These clones were used to from a set up in the HindIII site of the plasmid pUCl9 cDNA library from growing tubers of Solanum tuberosum clones full length (full-length clones). A clone with an insertion size 2.909 bp was for used the further cloning.

To produce the plasmid p35S-anti-BE, the cDNA insert was provided with the 35S-RNA promoter of the cauliflower mosaic virus (CaMV) and the polyadenylation signal of the octopine synthase of the Ti plasmid pTiACH5. The orientation of the cDNA was chosen so that the non-coding strand is read from the promoter (antisense orientation). The plasmid p35S-anti-BE has a size of 13.6 kb and consists of the three fragments A, B and C which are inserted into the polylinker of the plasmid pBIN19 (Bevan, Nucl. Acids Res. 12, 8711-8721) (s. 1 ).

Fragment A (529 bp) contains the CaMV promoter and includes nucleotides 6909 to 7437 (Franck et al., Cell 21: 285-294). It was generated as an EcoRI / KpnI fragment from the plasmid pDH51 (Pietrzak et al., Nucl. Acid Res. 14, 5857-5868) isolated and between the EcoRI / KpnI interfaces of the polylinker ligated from pBIN19. This resulted in pBIN19-A.

Fragment C (192 bp) contains this 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, which as a PvuII / HindIII fragment the plasmid pAGV40 (Herrera-Estrella et al., Nature 303, 209-213) was isolated and after adding SphI linkers to the PvuII interface between the SphI and HindIII sites of pBIN19-A were ligated. This resulted in pBIN19-AC.

Fragment B contains the 2909 bp cDNA of the branching enzyme from Solanum tuberosum and was isolated as a HindIII / SmaI fragment from the pUC19 derivative described above. After the HindIII site had been filled in using the DNA polymerase, the fragment was ligated into the SmaI site of the previously described derivative pBIN19-AC (see. 1 ).

Example 2

Construction of the plasmid p35SH anti-D

als Primer für eine Polymerase-Kettenreaktion an cDNA aus Knollengewebe von Solanum tuberosum wurde eine Kopie der Kodierregion des Disproportionierungsenzyms erzeugt, die wegen der spezifischen Sequenz der Oligonukleotide am 3'-Ende des kodoge nen Stranges mit einer Kpnl-Schnittstelle und an dessen 5'-Ende mit einer SmaI-Schnittstelle ausgestattet ist. Mit Hilfe dieser beiden Schnittstellen ist es möglich, die Kodierregion des Disproportionierungsenzyms in antisense-Orientierung zwischen die Kpnl- und die SmaI-Schnittstelle des binären Plasmids pBIN19-HYG zu klonieren. Das Plasmid pBIN19-HYG trägt das hphI-Gen für Hygromicinresistenz in der T-DNA. Die Verwendung dieses Plasmids ist notwendig, um Pflanzen, die bereits mit einem pBIN19-Derivat transformiert wurden, einer erneuten Transformation und Selektion unterziehen zu können. Für die Herstellung des Plasmids p35SH-anti-D wurde ein Derivat von pBIN19-HYG verwendet, das entsprechend der in Beispiel 1 beschriebenen Vorgehensweise mit den dort beschriebenen Fragmenten A und C ausgestattet worden war. Dies hatte zu dem Plasmid pBIN19-HYG-AC geführt.Using two synthetically produced DNA oligonucleotides of the sequences:

As a primer for a polymerase chain reaction on cDNA from tuber tissue from Solanum tuberosum, a copy of the coding region of the disproportionation enzyme was generated, which because of the specific sequence of the oligonucleotides at the 3'-end of the codogenic strand with a Kpnl site and at its 5'- End is equipped with an SmaI interface. With the help of these two interfaces it is possible to clone the coding region of the disproportionation enzyme in the antisense orientation between the Kpnl and the SmaI interface of the binary plasmid pBIN19-HYG. The plasmid pBIN19-HYG carries the hphI gene for hygromicin resistance in the T-DNA. The use of this plasmid is necessary in order to be able to subject plants which have already been transformed with a pBIN19 derivative to a new transformation and selection. For the preparation of the plasmid p35SH-anti-D, a derivative of pBIN19-HYG was used, which had been equipped with the fragments A and C described there in accordance with the procedure described in Example 1. This led to the plasmid pBIN19-HYG-AC.

The plasmid p35SH-anti-D was formed by ligation of the product of the polymerase chain reaction cut with Kpnl and SmaI to duplicate the coding region of the disproportionation enzyme between the Kpnl and SmaI interfaces of pBIN19-HYG-AC (see 2 ) which, like the plasmid p35S-anti-BE described in Example 1, consists of three fragments A, B and C, inserted into the polylinker of pBIN19-HYG-AC. Fragments A and C are identical to the corresponding ones in Example 1, fragment B contains nucleotides 1777 to 303 of the disproportionation enzyme from Solanum tuberosum (Takaha et al., 1993).

Example 3

Construction of the binary plasmid p35SH-anti-BE

Another clone, BE7, with an insertion of 3047 base pairs that code for the branching enzyme, as described in Example 1 from a cDNA library from potato tuber tissue isolated and into the expression vector λZAPII (Kossmann et al., Mol. Gene. Genet. 230, 39-44) inserted.

As described in Examples 1 and 2, this cDNA molecule was inserted as fragment B in the antisense orientation to the 355 promoter in the vector pBIN19-HYG-AC. The DNA region coding for the branching enzyme was isolated as a 3068 bp SmaI / HindIII fragment from a BE7 clone. After the protruding Hind III ends had been filled in using the Klenow fragment of DNA polymerase from Escherichia coli, the fragment was ligated into the SmaI site of the derivative pBIN19-HYG-AC described in Example 2. The resulting plasmid p35SH-anti-BE is in 3 shown.

Example 3

Construction of the binary plasmid p35SH-anti-BE

Another clone, BE7, with one for the branching enzyme encoding 3047 bp insert as in Example 1 described from a cDNA library from potato tuber tissue isolated and inserted into the expression vector λZAPII (Kossmann et al., Mol. Gen. Genet. 230, 39-44).

As described in Examples 1 and 2, this cDNA molecule was inserted as fragment B in the vector pBIN19-HYG-AC in antisense orientation to the 35S promoter. The cDNA encoding the branching enzyme was isolated from the BE7 clone as a 3068 bp SmaI / HindIII fragment. After the overlapping Hind III ends were filled in using the Klenow fragment of DNA polymerase I from Escherichia coli, the fragment was ligated into the SmaI site of the derivative pBIN19-HYG-AC described in Example 2. The resulting plasmid p35SH-anti-BE is shown in 3 shown.

Example 4

Construction of the binary plasmid p35SH anti-D

Analogously to Example 2, the PCR fragment described there, which codes for the disproportionation enzyme, was inserted as fragment B in the vector pBIN19-AC described in Example 1 in antisense orientation to the 35S promoter. The resulting plasmid p35S anti-D is shown in 4 shown.

Example 5

Construction of the binary plasmid p35S-anti-D-anti-BE

The insert described in Example 3, which codes for the branching enzyme, was inserted as a 3031 bp NotI fragment (fragment D) in the plasmid p35S-anti-D described in Example 4 in antisense orientation to the 35S promoter. After the NotI cleavage sites had been filled in using the Klenow fragment of DNA polymerase I, the fragment was ligated into plasmid p35S-anti-D cleaved with SmaI. The resulting plasmid p35S-anti-D-anti-BE is shown in 5 shown.

Example 6

Agrobacterium transformation tumefaciens with binary Plasmids and genetic modification of plants using transformed agrobacteria

The binary plasmids from the examples were used to transform Agrobacterium tumefaciens 1 to 5 into the cells by direct transformation according to the method of Höfgen & Willmitzer (1988, Nucl. Acids Res. 16, 9877). The plasmid DNA of transformed agrobacteria was determined by the method of Birnboim et al. (1979, Nucl. Acids Res. 7, 1513-1523) isolated and analyzed by gel electrophoresis after suitable restriction cleavage.

Agrobacteria, where the integrity of the in the embodiments 1 to 5 described binary Plasmids detected after transformation by restriction analysis had been used to genetically modify potato plants used.

To transform z. B. of potato plants For example, 10 small leaves wound with a scalpel become one Sterile culture in 10 ml MS medium with 2% sucrose, which 30–50 μl one under Selection of grown Agrobacterium overnight culture contains. After 3 - 5 minutes gentle shaking the Petri dishes at 25 ° C incubated in the dark. After 2 days, the leaves on MS medium with 1.6% Glucose, 2 mg / l zeatin ribose, 0.02 mg / l naphthylacetic acid, 0.02 mg / l giberellic acid, 500 mg / l Claforan, 50 mg / l kanamycin and 0.8% Bacto agar. To one week Incubation at 25 ° C and 3000 lux, the Claforan concentration in the medium is reduced by half. The further cultivation was carried out as described by Rocha-Sosa et al. (1989, EMBO J. 8, 29).

• a) Im ersten Fall wurden Kartoffelpflanzen zunächst mit dem in Beispiel 1 beschriebenen Plasmid transformiert, da der Erfolg der Antisense-Inhibition der Bildung des Verzweigungsenzym leicht mit dem in Beispiel 1 beschriebenen Antiserum vorgenommen werden kann. Transgene Pflanzen, bei denen kein Verzweigungsenzym mehr nachweisbar ist, werden für eine Superinfektion mit Plasmid p35SH-anti-D eingesetzt. Anstelle des Kanamycins wird nun zur Selektion Hygromycin in einer Konzentration von 3 mg/l verwendet.
• b) Analog zu dem unter a) beschriebenen Verfahren wurden Kartoffelpflanzen zunächst mit dem in Beispiel 3 beschriebenen Plasmid in einem reziproken Verfahren transformiert, und ein gegen das Disproportionierungsenzym gerichtetes Antiserum wurde verwendet, um Pflanzen zu identifizieren und auszuwählen, die eine stark verminderte Expression des Disproportionierungsenzym zeigen. Diese Pflanzen wurden dann mit dem in Beispiel 4 beschriebenen Plasmid superinfiziert.
• c) Alternativ werden Kartoffelpflanzen mit dem in Beispiel 5 beschriebenen Plasmid p35S-anti-D-anti-BE transformiert und Regeneranten ausgewählt, die einen stark verminderten Gehalt beider Proteine aufweisen.

The following three different methods were selected for introducing the DNA combination according to the invention into plants:

• a) In the first case, potato plants were first transformed with the plasmid described in Example 1, since the success of the antisense inhibition of the formation of the branching enzyme can easily be carried out with the antiserum described in Example 1. Transgenic plants in which no branching enzyme can be detected are used for superinfection with plasmid p35SH-anti-D. Instead of the kanamycin, hygromycin is now used in a concentration of 3 mg / l for the selection.
• b) Analogously to the method described under a), potato plants were first transformed with the plasmid described in Example 3 in a reciprocal process, and an antiserum directed against the disproportionation enzyme was used to identify and select plants which show a greatly reduced expression of the disproportionation enzyme demonstrate. These plants were then superinfected with the plasmid described in Example 4.
• c) Alternatively, potato plants are transformed with the plasmid p35S-anti-D-anti-BE described in Example 5 and regenerants are selected which have a greatly reduced content of both proteins.

The success of the genetic modification of the plants can be checked by analyzing the total RNA for the absence of the mRNA of the Q and D enzymes. Total RNA from plants was isolated according to Logemann et al. (1987, Anal. Biochem. 163, 16-20). For the analysis, 50 μg of the total RNA are examined for the absence of the Q and D enzyme transcripts using "Northern blots". The results of these "Northern Blot" analyzes are shown in the 6 . 7 and 8th shown. It is clear that the majority of the potato plants which have been transformed by the various methods with the DNA combination according to the invention have brought about a substantial reduction in the enzymes coding for the branching enzyme and the disproportionation enzyme.

To check the presence of the Q enzyme in transgenic plants, total protein was extracted from plant tissue and then "Western blot" analyzed with an appropriate antiserum. For this, protein extracts are separated by molecular weight using SDS-PAGE. After SDS-PAGE, protein gels are equilibrated for 15-30 minutes in transfer buffer for graphite electrodes (48 g / l Tris, 39 g / l glycine, 0.0375% SDS, 20% methanol) and then at 4 ° C. on a nitrocellulose -Filter transferred at 1.3 mA / cm ² for 1-2 hours. The filter is saturated for 30 minutes with 3% gelatin in TBS buffer (20 mM Tris / HCl, pH 7.5; 500 mM NaCl). The filter is then incubated for 2 hours with the antiserum in a suitable dilution (1: 1000-10,000 in TBS buffer) at room temperature. The filter is then washed for 15 minutes with TBS, TTBS (TBS buffer with 0.1% sorbitan monolaurate 20 EO) and TBS buffer. After waking, the filter is incubated for 1 hour at room temperature with goat anti-rabbit (GAR) antibodies conjugated to alkaline phosphatase (1: 7500 in TBS). The filter is then washed as described above and equilibrated in AP buffer (100 mM Tris / HCl, pH 9.5; 100 mM NaCl, 5 mM MgCl ₂ ). The alkaline phosphatase reaction is carried out by adding 70 μl of 4-nitrotetrazolium (NBT) solution (50 mg / ml NBT in 70% dimethylformamide) and 35 μl of 5-bromo-4-chloro-3-indolylphosphate (BCIP) (50 mg / ml BCIP in dimethylformamide) started in 50 ml AP buffer. The first signals can usually be observed after 5 minutes.

To determine the amylose / amylopectin content in the starch of transgenic potato plants that produce a starch with a different degree of branching, leaf pieces with a diameter of 10 mm are floated in 6% sucrose solution under constant light for 14 hours. This light incubation induces a greatly increased starch formation in the leaf pieces. After the incubation, the amylose and amylopectin concentration is determined according to Hovenkamp-Hermelink et al. (1988, Potato Research 31, 241-246).

The determination of the degree of branching (Content of α-1,6 bonds), the chain length and the size of the starch granules according to Morrison et al. (1990, Methods in Plant Biochemistry Academic Press Lmtd. 2, 323-352).

Claims (28)

Preparation containing a combination of DNA sequences consisting of A) the coding region of a branching enzyme or part of it, and B) the coding region of a disproportionation enzyme or part of it, each to a promoter in an antisense orientation are merged, and that after the introduction to a plant genome in transgenic plants results in the synthesis of transcripts, which is the synthesis of branching enzyme and disproportionation enzyme inhibit in the cells, whereby in the cells the synthesis of a modified Strength results, which differs naturally from that in starches synthesized in the cells in terms of their degree of branching and their phosphate content different. Preparation according to claim 1, characterized in that the coding regions defined in a) and b) of claim 1 or parts thereof are fused to a common promoter. Preparation according to claim 1, characterized in that the coding regions defined in a) and b) of claim 1 or parts thereof are each fused to their own promoter and therefore independent can be transcribed from each other. Preparation according to one of claims 1 to 3, characterized in that that the coding regions defined in a) or b) of claim 1 or parts thereof are located on a plasmid. Preparation according to claim 1 or 3, characterized in that that the coding regions defined in a) or b) of claim 1 or parts thereof are located on separate plasmids. Preparation according to one of claims 1 to 5, characterized in that that the coding regions defined in a) and b) of claim 1 or parts thereof come from Solanum tuberosum. Preparation according to claim 4, characterized in that the coding region of the branching enzyme and the coding region of the disproportionation enzyme on the plasmid p35S-anti-D-anti-BE (DSM 9367) are localized. Preparation according to claim 5 or 6, characterized in that that the coding region of the branching enzyme on the plasmid p35S-anti-BE (DSM 6144) and the coding region of the disproportionation enzyme the plasmid p35SH-anti-D (DSM 8479) are localized. Preparation according to claim 5 or 6, characterized in that that the coding region of the branching enzyme on the plasmid p35SH-anti-BE (DSM 9366) and the coding region of the disproportionation enzyme the plasmid p35S-anti-D (DSM 9365) are localized. Process for the production of transgenic plants which are able to form a modified starch, wherein first a DNA combination, as defined in one of claims 1 to 9, is stably integrated into the genome of a plant cell and then whole plants are transformed therefrom Plant cells are regenerated, characterized in that this method represents: A) a one-step method that comprises the following steps: a) production of a combination of DNA sequences from the following partial sequences: i) one promoter each, which is active in plants and the Ensures formation of an RNA in the intended target tissue or cells; ii) a coding region of a branching enzyme or a part thereof and a disproportionation enzyme or a part thereof, fused to the promoter mentioned under i) in such a way that a transcript is formed from the non-coding strand (antisense fusion) and iii) a third '-untranslated sequence which leads to termination of transcription and addition of poly-A residues to the 3' end of the RNA in plant cells, fused to the sequence mentioned under ii) in such a way that the 3 'untranslated sequence to the 3 'end of the non-coding strand mentioned under ii) follows; b) transfer and incorporation of the combinations of DNA sequences into a plant genome to generate transgenic plant cells and c) regeneration of intact, whole plants from the transformed plant cells, or B) a one-step process that comprises the following steps: a) production of a Combination of DNA sequences from the following partial sequences: i) a promoter which is active in plants and ensures the formation of an RNA in the intended target tissue or the target cells, ii) the fusion of the coding region of a branching enzyme or a part thereof and the coding region of a disproportionation enzyme or a part of it, fused to one another such that both coding regions are read in the same direction (sense or antisense), and fused to the promoter mentioned under i) in such a way that a transcript of the non-coding strand of the fusion (antisense fusion) is formed and iii) a 3 'untranslated sequence which is used in plant cells for Termination of the transcription and addition of poly-A residues to the 3 'end of the RNA leads, fused to the sequence mentioned under ii) in such a way that the 3' non-translated sequence joins the 3 'end of the non-coding mentioned under ii) Stranges connects, b) transfer and incorporation of the combinations of DNA sequences into a plant genome to generate transgenic plant cells, and c) regeneration of intact, whole plants from the transformed plant cells, or C) a two-step process in which a DNA Sequence consisting of the following partial sequences: iv) a promoter which is active in plants and ensures the formation of an RNA in the intended target tissue or the target cells, v) a coding region of a branching enzyme or a part thereof, such as those mentioned under iv) Promoter fuses that a transcript is formed from the non-coding strand (fantisense fusion), and vi) a 3 'untranslated sequence that is described in Pf leads to termination of transcription and addition of poly-A residues to the 3 'end of the RNA, fused to the sequence mentioned under v) in such a way that the 3' untranslated sequence joins the 3 'end of the under v) called non-coding strand, is transferred into the genome of a plant cell and incorporated, and from this way a whole plant is regenerated from the genetically modified plants, and then by repeated transformation into cells of this genetically modified plant a different DNA sequence which results from the The following partial sequences consist: vii) a promoter which is active in plants and ensures the formation of an RNA in the intended target tissue or cells, viii) a coding region of a disproportionation enzyme or a part thereof, fused to the promoter mentioned under vii) such that a Transcript is formed from the non-coding strand (antisense fusion), and ix) of a 3'-nontranslati first sequence, which leads to the termination of transcription and addition of poly-A residues to the 3 'end of the RNA in plant cells, fused to the sequence mentioned under viii) in such a way that the 3' untranslated sequence is linked to the 3 ' End of the non-coding strand mentioned under viii), is also transferred into the genome of a plant cell and is incorporated, and finally the plant cells transformed in this way, which contain both coding regions or parts thereof, are again regenerated to produce an entire plant, or D) a two-stage process as described under C), the coding region mentioned under v) not coding for a branching enzyme but for a disproportionation enzyme, and the coding region mentioned under viii) not for a disproportionation enzyme but for a branching enzyme. Method according to claim 10, characterized in that the one mentioned under i), iv) or vii) Promoter is the 35S Pro motor of the cauliflower mosaic virus. Method according to claim 10, characterized in that the one mentioned under i), iv) or vii) Promoter of the B33 promoter of the Solanum tuberosum patin gene is. Method according to claim 10 or 11, characterized in that in the case of process variants C) and D) the plasmids p35S-anti-BE (DSM 6144) and p35SH-anti-D (DSM 8479), or p35SH-anti-BE (DSM 9366) and p35S-anti-D (DSM 9365), or derivatives thereof which have the property for a branching enzyme or encode a disproportionation enzyme or parts thereof, for the introduction of the DNA combination can be used in the cells. Method according to claim 10 or 11, characterized in that in the case of process variant B) the plasmid p35S-anti-D-anti-BE (DSM 9367) or a derivative thereof which has the property for a branching enzyme and encode a disproportionation enzyme or parts thereof for the introduction the DNA combination is used in the cells. Plant cells containing a DNA sequence combination as in one of the claims 1 to 9 defined. Use of plant cells according to claim 15 for the production of plants which form a modified starch, that of the naturally starch synthesized in the cells in terms of their degree of branching and their phosphate content is different. Transgenic plant cells containing a DNA sequence combination as in one of the claims 1 to 9 defined. Transgenic plant according to claim 17, characterized in that it's a potato plant. Use of the DNA sequence combination as in one of claims 1 to 9 defined for the production of plant cells or plants, which is a modified strength form that of the naturally starch synthesized in the cells in terms of their degree of branching and their phosphate content is different. Use according to claim 19, characterized in that in a) or b) of claim 1 defined coding regions or parts thereof on separate plasmids lie and for simultaneous or sequential introduction to a plant genome can be used. Use according to claim 19, characterized in that in a) or b) of claim 1 defined coding regions or parts thereof on a plasmid lie. Use of the plasmids p35S-anti-BE (DSM 6144) and p35SH anti-D (DSM 8479), or p35S-anti-D (DSM 9365) and p35SH-anti-BE (DSM 9366), in combination, for the transformation of plant cells in order to Cells a modified strength to form that of the naturally starch synthesized in the cells in terms of their degree of branching and their phosphate content is different. Use of plasmid p35S-anti-D-anti-BE (DSM 9367) for the transformation of plant cells to a modified starch to form that of the naturally in starch synthesized in the cells in terms of their degree of branching and their phosphate content is different. Plasmid containing a DNA sequence combination, consisting of A) the coding region of a branching enzyme or part of it, and B) the coding region of a disproportionation enzyme or part of it, both in antisense orientation are fused to a promoter, the plasmid being introduced into a plant genome in transgenic plants for the formation of transcripts leads, which are the synthesis of branching enzyme and disproportionation enzyme inhibit in the cells so that in the cells the synthesis of a modified Strength which results from the naturally in starch synthesized in the cells in terms of their degree of branching and their phosphate content is different. A plasmid according to claim 24, characterized in that in a) or b) of claim 24 defined coding regions or parts thereof to a common one Promoter are fused. A plasmid according to claim 24, characterized in that in a) or b) of claim 24 defined coding regions or parts of each on their own promoter are fused and are therefore transcribed independently of each other can. A plasmid according to claim 24, characterized in that in a) or b) of claim 24 defined coding regions or parts thereof from Solanum tuberosum come. Plasmid p35S-anti-D-anti-BE (DSM 9367).