EP0904389A1 - Molecules d'acide nucleique codant des synthases d'amidon solubles obtenues a partir de mais - Google Patents

Molecules d'acide nucleique codant des synthases d'amidon solubles obtenues a partir de mais

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Publication number
EP0904389A1
EP0904389A1 EP97923925A EP97923925A EP0904389A1 EP 0904389 A1 EP0904389 A1 EP 0904389A1 EP 97923925 A EP97923925 A EP 97923925A EP 97923925 A EP97923925 A EP 97923925A EP 0904389 A1 EP0904389 A1 EP 0904389A1
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EP
European Patent Office
Prior art keywords
nucleic acid
starch
plant
gly
leu
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EP97923925A
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German (de)
English (en)
Inventor
Jens Kossmann
Claus Frohberg
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Bayer Bioscience GmbH
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Planttec Biotechnologie GmbH Forschung and Entwicklung
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Publication of EP0904389A1 publication Critical patent/EP0904389A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present invention relates to nucleic acid molecules that encode a form of soluble corn starch synthase. Furthermore, this invention relates to vectors, bacteria, and plant cells transformed with the nucleic acid molecules described, and plants regenerable therefrom. Furthermore, methods for the production of transgenic plants are described which, due to the introduction of DNA molecules which encode a soluble starch synthase from corn, synthesize a starch which has changed its properties.
  • the polysaccharide starch is a polymer made up of chemically uniform basic building blocks, the glucose molecules. However, this is a very complex mixture of different molecular forms which differ in their degree of polymerization and the occurrence of branches in the glucose chains. Starch is therefore not a uniform raw material.
  • amylose and Starch an essentially unbranched polymer of ⁇ -1,4-glycosidically linked glucose molecules, of the amylopectin starch, which in turn is a complex mixture of differently branched glucose chains.
  • the branches come about through the occurrence of additional ⁇ -1,6-glycosidic linkages.
  • the synthesized starch consists of approx.
  • the biochemical synthetic routes that lead to the build-up of starch are essentially known.
  • the starch synthesis in plant cells takes place in the plastids.
  • photosynthetically active tissues these are the chloroplasts, in photosynthetically inactive, starch-storing tissues, the amyloplasts.
  • starch synthases The most important enzymes involved in starch synthesis are starch synthases and branching enzymes. Various isoforms are described for starch synthases, all of which catalyze a polymerization reaction by transferring a glucosyl residue from ADP-glucose to ⁇ -1,4-glucans. Branching enzymes catalyze the introduction of ⁇ -1,6-branches into linear ⁇ -1,4-glucans.
  • Starch synthases can be divided into two classes: the starch-grain-bound starch synthases ("granule-bound starch synthases”; GBSS ⁇ and the soluble starch synthases ("soluble starch synthases”; SSS). This distinction cannot be clearly made in each case because some of the starch synthases 2> are both starch-bound and in soluble form (Denyer et al., Plant J. 4 (1993), 191-198; Mu et al., Plant J. 6 (1994), 151-159). Within these classes, different isoforms are described for different plant species, which differ in terms of their dependence on starter molecules (so-called “primer dependent” (type II) and “primer independent” (type I) starch synthases).
  • GBSS I is not only involved in the synthesis of amylose, but also has an influence on amylopectin synthesis.
  • mutants that have no GBSS I activity a certain fraction of the normally synthesized amylopectin, which has longer-chain glucans, is missing.
  • Gen Genet 203 (1986) 237-244) has been described (Shen et al, 1994, GenBank No. T14684), the derived amino acid sequence of which is very similar to the derived ammosaic sequence of GBSS II from pea (Dry et al., Plant J 2 (1992), 193-202) and » (Edwards et al., Plant J 8 (1995), 283-294) has nuclear acid sequences which code for further starch synthase isoforms from maize, however, have not yet been available.
  • cDNA sequences coding for starch synthases other than GBSS I have so far only been used for pea (Dry et al., Plant J. 2 (1992), 193-202), rice (Baba et al., Plant Physiol 103 ( 1993), 565-573) and potato (Edwards et al., Plant J. 8 (1995), 283-294)
  • soluble starch synthases have also been identified in a number of other plant species. Soluble starch synthases are, for example, homogeneous from pea (Denyer and Smith, Planta 186 (1992), 609-617) and potato (Edwards et al., Plant J 8 (1995), 283-294) In these cases it was found that the isoform of soluble starch synthase identified as SSS II is identical to the starch synthase-bound starch synthase GBSS II (Denyer et al., Plant J. 4 ( 1993), 191-198; Edwards et al., Plant J. 8 (1995), 283-294).
  • the present invention is therefore based on the object of making available nucleic acid molecules which encode enzymes involved in starch biosynthesis and with them s
  • the present invention therefore relates to nucleic acid molecules which encode proteins with the biological activity of a soluble starch synthase of type I from maize, such molecules preferably encoding proteins which have the Seq ID no. 2 given amino acid sequence include.
  • the invention relates to nucleic acid molecules which are listed under Seq ID No. 1 or a part thereof, preferably molecules which contain the nucleotide sequence specified in Seq ID No. 1 indicated coding region comprise or corresponding ribonucleotide sequences.
  • the present invention further relates to nucleic acid molecules which encode a soluble starch synthase from maize and whose one strand hybridizes with one of the molecules described above or with a complementary strand of these molecules.
  • the invention also relates to nucleic acid molecules which encode a soluble starch synthase of type I from maize and whose sequence deviates from the nucleotide sequences of the molecules described above due to the degeneration of the genetic code.
  • the invention also relates to nucleic acid molecules which have a sequence which is complementary to all or part of the sequence of the abovementioned molecules.
  • the nucleic acid molecules according to the invention can be both DNA and RNA molecules.
  • Corresponding DNA molecules are, for example, genomic or cDNA molecules.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrock et al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention can originate from any maize plant which has such molecules.
  • Nucleic acid molecules that hybridize with the molecules according to the invention can be isolated, for example, from genomic or from cDNA libraries of maize plants or maize plant tissue. Alternatively, they can be produced by genetic engineering methods or by chemical synthesis.
  • nucleic acid molecules can be identified and isolated using the molecules according to the invention or parts of these molecules or the reverse complements of these molecules, e.g. by means of hybridization according to standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • hybridization sample e.g. Nucleic acid molecules are used which exactly or essentially those under Seq ID No. 1 indicated nucleotide sequence or parts of this sequence.
  • the fragments used as the hybridization sample can also be synthetic fragments which were produced with the aid of the usual synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention. If genes which hybridize with the nucleic acid sequences according to the invention have been identified and isolated, a determination of the sequence and an analysis of the properties of the proteins coded by this sequence are necessary.
  • the molecules hybridizing with the nucleic acid molecules according to the invention also include fragments, derivatives and all variants of the nucleic acid molecules described above, which encode a soluble starch synthase according to the invention from maize. Fragments are understood to mean parts of the nucleic acid molecules that are long enough to encode one of the proteins described.
  • the term derivative in this context means that the sequences of these molecules are different differ from the sequences of the nucleic acid molecules described above at one or more positions and have a high degree of homology to these sequences.
  • 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 from the nucleic acid molecules described above may have resulted from deletion, substitution, insertion or recombination.
  • nucleic acid molecules in question or the proteins encoded by them are usually variations of these molecules which are modifications which have the same biological function.
  • These can be both naturally occurring variations, for example sequences from other maize varieties, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis.
  • the variations can be synthetically produced sequences.
  • allelic variants can be both naturally occurring variants and synthetically produced variants or those produced by recombinant DNA techniques.
  • the proteins encoded by the different variants of the nucleic acid molecules according to the invention have certain common characteristics. For this, e.g. Enzyme activity, molecular weight, immunological reactivity, conformation etc. include, as well as physical properties such as the running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficient, solubility, spectroscopic properties, stability; pH optimum, temperature optimum, etc.
  • a starch synthase Important characteristics of a starch synthase are: i) its localization in the stroma of plastids in plant cells; ii) their ability to synthesize linear ⁇ -1,4 linked polyglucans using ADP-glucose as a substrate. This Q
  • the proteins encoded by the nucleic acid molecules according to the invention are a previously unidentified and characterized form of a soluble starch synthase from maize, which can be assigned to type I ("primer independent"). Such starch synthases or nucleic acid molecules which encode such proteins have not previously been described in maize.
  • the encoded protein has a certain homology to a soluble starch synthase from rice (Baba et al., Plant Physiol. 103 (1993), 565-573).
  • the invention also relates to oligonucleotides which hybridize specifically with a nucleic acid molecule according to the invention.
  • Such oligonucleotides preferably have a length of at least 10, in particular at least 15 and particularly preferably at least 50 nucleotides. They are characterized in that they hybridize specifically with nucleic acid molecules according to the invention, i.e. not or only to a very small extent with nucleic acid sequences that encode other proteins, in particular other starch synthases.
  • the oligonucleotides according to the invention can be used, for example, as primers for a PCR reaction. They can also be components of antisense constructs or of DNA molecules which code for suitable ribozymes.
  • the invention further relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above.
  • the nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure the transcription and synthesis of a translatable RNA in prokaryotic or eukaryotic cells.
  • nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, is interesting in that it is more accurate in this way 3
  • nucleic acid molecules according to the invention which results in the synthesis of proteins with possibly changed biological properties.
  • deletion mutants in which nucleic acid molecules are generated by progressive deletions from the 5 'or 3' end of the coding DNA sequence, which lead to the synthesis of correspondingly shortened proteins.
  • deletions at the 5 'end of the nucleotide sequence make it possible, for example, to identify amino acid sequences which are responsible for the translocation of the enzyme into the plastids (transit peptides). This makes it possible to specifically produce enzymes which are no longer localized in the plastids but instead in the cytosol by removing the corresponding sequences, or which are localized in other compartments due to the addition of other signal sequences.
  • the introduction of point mutations is also conceivable at positions in which a change in the amino acid sequence has an influence, for example on the enzyme activity or the regulation of the enzyme. In this way it is possible, for example, to produce mutants which have a changed K m value or which are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification.
  • mutants can be produced which have a changed substrate or product specificity, such as, for example, mutants which use ADP-glucose-6-phosphate as the substrate instead of ADP-glucose. Mutants can also be produced Those who have a changed activity-temperature profile.
  • the nucleic acid molecules according to the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or a sequence change by recombination of DNA sequences.
  • base exchanges can be carried out or natural or synthetic sequences can be added.
  • adapters or linkers can be attached to the fragments.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and other biochemical-molecular-biological methods are generally carried out as the analysis method.
  • the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which are transformed with a nucleic acid molecule according to the invention or a vector according to the invention described above, and cells which are derived from cells transformed in this way and contain a nucleic acid molecule or a vector according to the invention.
  • host cells in particular prokaryotic or eukaryotic cells, which are transformed with a nucleic acid molecule according to the invention or a vector according to the invention described above, and cells which are derived from cells transformed in this way and contain a nucleic acid molecule or a vector according to the invention.
  • host cells in particular prokaryotic or eukaryotic cells, which are transformed with a nucleic acid molecule according to the invention or a vector according to the invention described above, and cells which are derived from cells transformed in this way and contain a nucleic acid molecule or a vector according to the invention.
  • These are preferably bacterial cells or plant cells.
  • the invention further relates to the proteins which are encoded by the nucleic acid molecules according to the invention, and to processes for their production, a host cell according to the invention being cultivated under conditions which allow the synthesis of the protein, and then the protein from the cultivated cells and / or the culture medium is isolated.
  • the present invention thus also relates to transgenic plant cells which transform with a nucleic acid molecule according to the invention, i.e. were genetically modified, as well as transgenic plant cells derived from such transformed cells and containing nucleic acid molecules according to the invention.
  • nucleic acid molecules according to the invention it is now possible, using genetic engineering methods, to intervene in the starch metabolism of plants, as was previously not possible, and to change it in such a way that a modified starch is synthesized, for example in its physicochemical properties, in particular the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the starch grain size and / or the starch grain shape is changed compared to starch synthesized in wild-type plants.
  • a modified starch for example in its physicochemical properties, in particular the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the starch grain size and / or the starch grain shape is changed compared to starch synthesized in wild-type plants.
  • nucleic acid molecules according to the invention by methods known to the person skilled in the art in order to obtain starch synthases according to the invention which are no longer subject to the cell's own regulatory mechanisms or which have changed temperature dependencies or substrate or product specificities.
  • the cells according to the invention contain one according to the invention
  • Nucleic acid molecule this being preferably linked to regulatory DNA elements which ensure transcription in plant cells, in particular to a promoter.
  • Such cells can be distinguished from naturally occurring plant cells in that they contain a nucleic acid molecule according to the invention which does not naturally occur in these cells or in that such a molecule is integrated at a location in the genome of the cell where it does not otherwise occur , ie in a different genomic environment.
  • the nucleic acid molecules according to the invention are expressed in plants, there is in principle the possibility that the synthesized protein can be localized in any compartment of the plant cell.
  • sequence ensuring localization in plastids must be deleted and the remaining coding region may have to be linked to DNA sequences which ensure localization in the respective compartment.
  • sequences are known (see for example Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al ., Plant J. 1 (1991), 95-106).
  • the present invention also relates to plants which contain cells according to the invention. These can be obtained, for example, by regeneration of the transgenic plant cells according to the invention using methods known to the person skilled in the art.
  • the transgenic plants can in principle be plants of any plant species, i.e. both monocot and dicot plants. They are preferably useful plants, in particular starch-synthesizing or starch-storing plants, such as e.g. Cereals (rye, barley oats, wheat etc.), rice, corn, peas, cassava or potatoes.
  • the invention also relates to propagation material of the plants according to the invention which contains cells according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, callus cultures, cell cultures etc.
  • the present invention also relates to the starch obtainable from the transgenic plant cells, plants and propagation material according to the invention.
  • the transgenic plant cells and plants according to the invention synthesize a starch which has, for example, its physicochemical properties, in particular the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the Gelatinization behavior, the starch grain size and / or the starch grain shape is changed in comparison to starch synthesized in wild type plants.
  • a starch can be changed with regard to the viscosity and / or the gel formation properties of paste of this starch compared to wild-type starch.
  • Another object of the invention are transgenic maize plant cells in which the activity of a protein according to the invention is reduced compared to non-transformed cells.
  • nucleic acid molecules according to the invention With the aid of the nucleic acid molecules according to the invention, it is possible to produce maize plant cells and maize plants in which the activity of a protein according to the invention is reduced. This also leads to the synthesis of a starch with changed chemical and / or physical properties compared to starch from wild-type plant cells.
  • the production of maize plant cells with a reduced activity of a protein according to the invention can be achieved, for example, by the expression of a corresponding antisense RNA, a sense RNA to achieve a cosuppression effect or the expression of a correspondingly constructed ribozyme which specifically cleaves transcripts which encode one of the proteins according to the invention using the nucleic acid molecules according to the invention.
  • ribozymes for reducing the activity of certain enzymes in cells is also known to the person skilled in the art and is described, for example, in EP-Bl 0 321 201.
  • the expression of ribozymes in plant cells was described, for example, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
  • an antisense RNA is preferably expressed in plant cells.
  • a DNA molecule can be used that comprises the entire sequence coding for a protein according to the invention, including any flanking sequences that may be present, as well as DNA molecules that only comprise parts of the coding sequence, these parts having to be long enough to be able to to cause the cells an antisense effect.
  • sequences up to a minimum length of 15 bp, preferably a length of 100-500 bp can be used for an efficient antisense inhibition, in particular sequences with a length of more than 500 bp.
  • DNA molecules are used that are shorter than 5000 bp, preferably sequences that are shorter than 2500 bp.
  • DNA sequences which have a high degree of homology to the sequences of the DNA molecules according to the invention, but which are not completely identical.
  • the minimum homology should be greater than approximately 65%.
  • sequences with homologies between 95 and 100% is preferred.
  • the invention also relates to maize plants which contain transgenic maize plant cells according to the invention.
  • the invention also relates to propagation material of the plants according to the invention, in particular seeds.
  • the invention also relates to the starch obtainable from the transgenic maize plant cells, maize plants and propagation material described above.
  • the transgenic maize plant cells and maize plants synthesize a starch which has, for example, its physicochemical properties, in particular the amylose / amylopectin ratio, the degree of branching, the average chain length, the Phosphate content, the gelatinization behavior, the starch grain size and / or the starch grain shape is changed compared to starch synthesized in wild-type plants.
  • This starch can show, for example, changed viscosities and / or gelation properties of its paste in comparison to starch from wild-type plants.
  • starches according to the invention can be modified by methods known to those skilled in the art and are suitable in unmodified or modified form for various uses in the food or non-food sector.
  • the use of starch can be divided into two large areas.
  • One area comprises the hydrolysis products of starch, mainly glucose and glucan building blocks, which are obtained via enzymatic or chemical processes. They serve as the starting material for further chemical modifications and processes, such as fermentation.
  • the simplicity and cost-effective implementation of a hydrolysis process can be important for reducing the costs.
  • it is essentially enzymatic using amyloglucosidase. It would be conceivable to save costs by using fewer enzymes.
  • a structural change in strength e.g. This could result in an increase in the surface area of the grain, easier digestibility due to a lower degree of branching or a steric structure which limits the accessibility for the enzymes used.
  • Starch is a classic additive for many foodstuffs, in which it essentially takes on the function of binding aqueous additives or causes an increase in viscosity or increased gel formation. Important characteristics are the flow and sorption behavior, the swelling and gelatinization temperature, the viscosity and thickening performance, the solubility of the starch, the transparency and paste structure, the heat, shear and acid stability, the tendency to retrogradation, the ability for film formation, freeze / thaw stability, digestibility and the ability to form complexes with, for example inorganic or organic ions.
  • the starch can be used as an auxiliary for different manufacturing processes or as an additive in technical products.
  • starch When using starch as an auxiliary, the paper and cardboard industry should be mentioned in particular.
  • the starch primarily serves for retardation (retention of solids), the setting of filler and fine particles, as a strengthening agent and for drainage.
  • the favorable properties of the starch in terms of rigidity, hardness, sound, feel, gloss, smoothness, splitting resistance and surfaces are used.
  • the requirements on starch with regard to the Oberflä ⁇ chen accent are essentially a high degree of brightness, corresponding viscosity, a high Viskosticiansstabili- ty, good film formation as well as low Staubbil ⁇ dung.
  • the solid content, an adapted viscosity, a high binding capacity and a high pigment affinity play an important role.
  • a rapid, uniform, loss-free distribution, high mechanical stability and complete restraint in the paper flow are important.
  • an adapted solids content, high viscosity and high binding capacity are also important.
  • starches A large area of use of the starches is in the adhesive industry, where the possible uses are divided into four areas: use as pure starch glue, use with starch glues prepared with special chemicals, use of starch as an additive to synthetic resins and polymer dispersions, and use of starches as an extender for synthetic adhesives.
  • 90% of the starch-based adhesives are used in the fields of corrugated cardboard manufacture, manufacture of paper sacks, bags and pouches, manufacture of composite materials for paper and aluminum, manufacture of cardboard packaging and rewetting glue for envelopes, stamps etc.
  • a large area of application for the strengths as an auxiliary and additive is the area of manufacture of textiles and textile care products.
  • the following four areas of application can be distinguished within the textile industry: the use of starch as a sizing agent, that is to say as an auxiliary for smoothing and strengthening the hook-and-loop behavior to protect against the tensile forces acting during weaving and for increasing the abrasion resistance during weaving, starch as a means of textile finishing especially after pre-treatments that deteriorate quality, such as bleaching, dyeing, etc., starch as a thickening agent in the manufacture of color pastes to prevent dye diffusion, and starch as an additive to chaining agents for sewing threads.
  • the fourth area of application is the use of starches as an additive in building materials.
  • One example is the production of gypsum plasterboard, in which the starch mixed in the gypsum slurry gelatinizes with the water, diffuses to the surface of the gypsum board and binds the cardboard to the board there. Further areas of application are admixing to plaster and mineral fibers. In ready-mixed concrete, starch products are used to delay the setting.
  • starch Another market for starch is in the manufacture of soil stabilizers that are used to temporarily protect soil particles from water during artificial earthmoving. Combination products made from starch and polymer emulsions are, according to current knowledge, equivalent in their erosion and incrustation-reducing effect to the products used hitherto, but are significantly lower in price.
  • starch in crop protection agents to change the specific properties of the preparations.
  • the starch can be used to improve the wetting of crop protection agents and fertilizers, for the metered release of the active substances, for converting liquid, volatile and / or malodorous substances into microcrystalline, stable, moldable substances, for mixing incompatible compounds and to extend the duration of action by reducing the decomposition.
  • starch can be used as a binder for tablets or for binder dilution in capsules. »9 sets.
  • the starch can furthermore serve as a tablet disintegrant, since after swallowing it absorbs liquid and swells to such an extent after a short time that the active substance is released.
  • Medical lubricant and wound powders are based on starch for qualitative reasons.
  • starches are used, for example, as carriers for powder additives such as fragrances and salicylic acid.
  • a relatively large area of application for starch is toothpaste.
  • Starch is used as an additive to coal and briquette. Coal can be agglomerated or briquetted with a high-quality addition of starch, thereby preventing the briquettes from disintegrating prematurely.
  • the starch addition is between 4 and 6% for barbecued coal and between 0.1 and 0.5% for calorized coal. Furthermore, starches are becoming increasingly important as binders, since their addition to coal and briquette can significantly reduce the emissions of harmful substances.
  • the starch can also be used as a flocculant in ore and coal sludge processing.
  • Another area of application is as an additive to casting auxiliaries.
  • Various casting processes require cores that are made from binder-mixed sands. Bentonite, which is mixed with modified starches, mostly swelling starches, is predominantly used today as a binder.
  • starch addition is to increase the flow resistance and to improve the binding strength.
  • swelling starches can have other production requirements, such as dispersible in cold water, rehydratable, good miscibility in sand and high water-binding capacity.
  • the starch can be used to improve the technical and optical quality.
  • the reasons for this are the improvement of the surface gloss, the improvement of the handle and the appearance, for this reason starch is sprinkled on the sticky rubberized surfaces of rubber materials before the cold vulcanization, and the improvement of the printability of the rubber.
  • Another way of selling the modified starches is in the production of leather substitutes.
  • starch secondary products in the processing process (starch is only filler, there is no direct link between synthetic polymer and starch) or, alternatively, the integration of starch secondary products in the production of polymers (starch and polymer form a firm bond).
  • starch as a pure filler is not competitive compared to other substances such as talc. It looks different if the specific starch properties come into play and the property profile of the end products is thereby significantly changed.
  • An example of this is the use of starch products in the processing of thermoplastics, such as polyethylene.
  • starch and the synthetic polymer are combined by coexpression in the ratio of 1: 1 combined to form a 'master batch 1, from the liertem with granulocytes polyethylene using conventional procedural ⁇ renstechniken various products are produced.
  • the integration of starch in polyethylene films an increased substance permeability in hollow bodies, improved water vapor permeability can ⁇ , improved antistatic behavior, improved anti-block behavior as well as improved printability Be ⁇ be achieved with aqueous dyes. 2 ⁇
  • starch in polyurethane foams.
  • starch derivatives By adapting the starch derivatives and by optimizing the process, it is possible to control the reaction between synthetic polymers and the hydroxyl groups of the starches.
  • the result is polyurethane films which, through the use of starch, obtain the following property profiles: a reduction in the coefficient of thermal expansion, reduction in shrinkage behavior, improvement in pressure / stress behavior, increase in water vapor permeability without changing the water absorption, reduction in flammability and tear density , no dripping of flammable parts, freedom from halogen and reduced aging.
  • Disadvantages that are currently still present are reduced compressive strength and reduced impact resistance.
  • Solid plastic products such as pots, plates and bowls can also be manufactured with a starch content of over 50%.
  • starch / polymer mixtures are to be judged favorably, since they have a much higher biodegradability.
  • starch graft polymers Because of their extreme water-binding capacity, starch graft polymers have also become extremely important. These are products with a backbone made of starch and a side grid of a synthetic monomer grafted on according to the principle of the radical chain mechanism.
  • the starch graft polymers available today are characterized by better binding and retention properties of up to 1000 g of water per g of starch with high viscosity.
  • the areas of application for these superabsorbents have expanded considerably in recent years and are in the hygiene sector with products such as diapers and pads as well as in the agricultural sector, e.g. seed pilling.
  • modified starches by means of genetic engineering interventions in a transgenic plant can on the one hand change the properties of the starch obtained from the plant in such a way that further modifications by means of chemical or physical processes no longer appear to be necessary.
  • the starches modified by genetic engineering processes can be subjected to further chemical modifications, which leads to further improvements in quality for certain of the fields of application described above.
  • nucleic acid molecules according to the invention in sense or antisense orientation in plant cells, which is linked to regulatory DNA elements that ensure transcription in plant cells.
  • regulatory DNA elements that ensure transcription in plant cells.
  • These include promoters in particular.
  • any promoter active in plant cells is suitable for expression.
  • the promoter can be selected so that the expression is constitutive or only in a certain tissue, at a certain time in plant development or at a time determined by external influences.
  • the promoter can be homologous or heterologous to the plant.
  • Suitable promoters for constitutive expression are, for example, the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize, for tuber-specific expression in potatoes the patatin gene promoter B33 (Rochasosa et al., EMBO J. 8 (1989 ), 23-29) or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LSI promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947 ; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or for endosperm-specific expression, the HMG promoter from wheat, the USP promoter, the phaseolin promoter or promoters of zein genes from maize.
  • the present invention provides nucleic acid molecules which encode a new form of a soluble starch synthase identified in maize. This now allows both the identification of the function of this starch synthase in starch biosynthesis and the production of genetically modified plants in which the activity of this enzyme is changed. This enables the synthesis of a starch with a changed structure and thus changed physicochemical properties in plants manipulated in this way.
  • the nucleic acid molecules according to the invention can also be used to produce plants in which the activity of the starch synthase according to the invention increases or is reduced and at the same time the activities of other enzymes involved in starch biosynthesis are changed. All combinations and permutations are conceivable.
  • the change in the activities of one or more isoforms of the starch synthases in plants leads to the synthesis of a starch which has a different structure.
  • Increasing the activity of one or more isoforms of the starch synthases in the cells of the starch-storing tissue of transformed plants, for example in the endosperm of maize or wheat or in the tuber of the potato, can also increase the yield.
  • nucleic acid molecules which code for a protein according to the invention or corresponding antisense constructs can be introduced into plant cells in which the synthesis of endogenous GBSS I, SSS or GBSS II proteins is already inhibited due to an antisense effect or a mutation or the synthesis of the branching enzyme is inhibited (as described, for example, in W092 / 14827 or the ae mutant (Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25-86)).
  • DNA molecules can be used for the transformation which simultaneously have several regions coding for the corresponding starch synthases in antisense orientation under the control of a suitable promoter contain.
  • each sequence can be under the control of its own promoter, or the sequences can be transcribed as a fusion from a common promoter. The latter alternative will generally be preferable, since in this case the synthesis of the corresponding proteins should be inhibited to approximately the same extent.
  • molecules which, in addition to sequences coding for starch synthases, contain further DNA sequences which code for other proteins involved in starch synthesis or modification. These are each coupled in an antisense orientation to a suitable promoter.
  • the sequences can in turn be connected in series and can be transcribed by a common promoter or else can be transcribed by separate promoters.
  • the length of the individual coding regions that are used in such a construct applies to what has already been stated above for the production of antisense constructs. There is no upper limit on the number of antisense fragments transcribed from a promoter in such a DNA molecule. However, the resulting transcript should generally not exceed a length of 10 kb, preferably 5 kb.
  • Coding regions which are located in such DNA molecules in combination with other coding regions in antisense orientation behind a suitable promoter can originate from DNA sequences which code for the following proteins: starch-grain-bound (GBSS I and II) and soluble starch synthases (SSS I and II), branching enzymes, "debranching" enzymes, disproportionation enzymes and starch phosphorylases. This is only an example. The use of other DNA sequences in the context of such a combination is also conceivable.
  • the constructs can furthermore be introduced into classic mutants which are defective for one or more genes of starch biosynthesis (Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London , 2nd Edition: 25-86). These defects can relate to the following proteins: starch-bound (GBSS I and II) and soluble starch synthases (SSS I and II), branching enzymes (BE I and II), "debranching" enzymes (R-enzymes), disproportionation enzymes and starch phosphorylases. This is only an example.
  • a large number of cloning vectors are available to prepare the introduction of foreign genes into higher plants, which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the desired sequence can be inserted into the vector at a suitable restriction site.
  • the plasmid obtained is used for the transformation of E. coli cells.
  • Transformed E. coli cells are grown in a suitable medium, then harvested and lysed. The plasmid is recovered.
  • Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained.
  • the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.
  • Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • a large number of techniques are available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and other possibilities.
  • plasmids When DNA is injected and electroporated into plant cells, there are no special requirements for the plasmids used. Simple plasmids such as e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, a selectable marker gene should advantageously be present.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but advantageously the right and left boundary of the Ti and Ri plasmid T-DNA should be connected as a flank region to the genes to be introduced.
  • the DNA to be introduced should be cloned into special plasmids, either in an intermediate vector or in a binary vector.
  • the intermediate vectors can be due to Sequences that are homologous to sequences in the T-DNA can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and agrobacteria.
  • the agrobacterium serving as the host cell should contain a plasmid carrying a vir region.
  • the vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • the agrobacterium transformed in this way is used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij 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.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells .
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • suitable medium which may contain antibiotics or biocides for the selection of transformed cells
  • EP 292 435 describes a method by means of which fertile plants can be obtained starting from a slimy, soft (friable) granular corn callus.
  • Shillito et al. Bio / Technology 7 (1989), 581) have observed in this connection that for the regenerability to fertile plants it is also necessary to start from callus suspension cultures from which a dividing protoplast culture, with the ability to regenerate plants, can be produced. After an in vitro cultivation time of 7 to 8 months, Shillito et al.
  • the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and remains in the offspring of the originally transformed cell. It usually contains a selection marker, the resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin, among others, is imparted to the transformed plant cells.
  • the individually selected marker should therefore allow the selection of transformed cells over cells which lack the inserted DNA.
  • the transformed cells grow within the plant in the usual way (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84).
  • the resulting plants can be grown normally and crossed with plants which have the same transformed genetic makeup or other genetic makeup.
  • the resulting hybrid individuals have the corresponding phenotypic properties. Seeds can be obtained from the plant cells.
  • Two or more generations should be attracted to ensure that the phenotypic trait is stable and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.
  • Figure 1 schematically shows the vector pUBIbar.
  • FIG. 2 schematically shows the vector pUBI-bar-aMasy
  • Ubiquitin-Pro Ubiquitin promoter
  • tumefaciens 35S 35S promoter of the CaMV
  • T35S 35S terminator of the CaMV
  • This vector contains, in antisense orientation to the ubiquitin promoter, the cDNA described in Example 1, which encodes a corn starch synthase. 2 > o
  • Protoplast isolation medium (100 ml)
  • Bovine Serum Albumin 20 mg
  • Protoplast washing solution 1 like protoplast isolation solution, but without cellulase, pectolyase and BSA
  • PEG 6000 is added to the above buffer under b) shortly before use of the solution (40% by weight PEG).
  • the solution is filtered through a 0.45 ⁇ m sterile filter.
  • Protoplast culture medium (in mg / 1)
  • the vector pBluescript II SK (Stratagene) was used for cloning in E. coli.
  • E.coli strain DH5 ⁇ (Bethesda Research Laboratories, Gaithersburgh, USA) was used for the Bluescript vector and for the pUSP constructs.
  • the E.coli strain XLl-Blue was used for in vivo excision.
  • the suspension is sieved through a stainless steel and nylon sieve with mesh sizes of 200 and 45 ⁇ m, respectively.
  • the combination of a 100 ⁇ m and a 60 ⁇ m sieve enables the cell aggregates to be separated just as well.
  • the filtrate-containing filtrate is assessed microscopically. It usually contains 98-99% protoplasts. The rest are undigested single cells.
  • Protoplast preparations with this degree of purity are used for transformation experiments without additional gradient centrifugation.
  • the protoplasts are sedimented by centrifugation (100 rpm in the oscillating rotor (100 ⁇ g, 3 min). The supernatant is discarded and the protoplasts are resuspended in washing solution 1. The centrifugation is repeated and the protoplasts are then resuspended in the transformation buffer.
  • the protoplasts resuspended in transformation buffer are filled into 50 ml polyallomer tubes with a titer of 0.5-1 ⁇ 10 protoplasts / ml in 10 ml portions.
  • the DNA used for the transformation is dissolved in Tris-EDTA (TE) buffer. 20 ⁇ g of plasmid DNA are added per ml of protoplast suspension. A plasmid imparting resistance to phosphinotricin is used as the vector (cf., for example, EP 0 513 849).
  • TE Tris-EDTA
  • a plasmid imparting resistance to phosphinotricin is used as the vector (cf., for example, EP 0 513 849).
  • the protoplast suspension is carefully shaken in order to distribute the DNA homogeneously in the solution. Immediately afterwards, 5 ml of PEG solution is added dropwise.
  • the PEG solution is distributed homogeneously by carefully swirling the tubes. Then another 5 ml of PEG solution are added and the homogeneous mixing is repeated. The protoplasts remain at ⁇ 2 ° C for 20 min of the PEG solution. The protoplasts are then passed through Centrifuged for 3 minutes (100 g; 1000 rpm). The supernatant is discarded. The protoplasts are washed by shaking gently in 20 ml of W5 solution and then centrifuged again. Then they are resuspended in 20 ml protoplast culture medium, centrifuged again and resuspended in culture medium.
  • the titer is set to 6 - 8 x 10 5 protoplasts / ml and the protoplasts are cultivated in 3 ml portions in petri dishes (0 60 mm, height 15 mm).
  • the petri dishes sealed with Parafilm are set up in the dark at 25 ⁇ 2 ° C.
  • the protoplasts are cultivated without the addition of fresh medium. As soon as the cells regenerated from the protoplasts have developed into cell aggregates with more than 20-50 cells, 1 ml of fresh protoplast culture medium which contains sucrose as an osmoticum (90 g / l) is added.
  • the cell aggregates formed from protoplasts can be plated on agar media with 100 mg / 1 L-phosphinothricin.
  • N6 medium with the vitamins of the protoplast culture medium, 90 g / 1 sucrose and 1.0 mg / 1 2, 4D is just as suitable as an analog medium, for example with the macro and micronutrient salts of the MS medium (Murashige and Skoog (1962 ) , see above) .
  • the calli resulting from stably transformed protoplasts can continue to grow unhindered on the selective medium.
  • the transgenic calli can be transferred to fresh selection medium, which is also 100 IS contains mg / 1 L-phosphinothricin, but no longer contains auxin.
  • fresh selection medium which is also 100 IS contains mg / 1 L-phosphinothricin, but no longer contains auxin.
  • the embryogenic transformed maize tissue is cultivated on hormone-free N6 medium (Chu CC et al., Sei. Sin. 16 (1975), 659) in the presence of 5 ⁇ 10 4 M L-phosphinothricin.
  • Maize embryos develop on this medium plants that express the phsphinothricin acetyltransferase gene (PAT gene) sufficiently strongly, untransformed embryos or those with only very weak PAT activity die as soon as the leaves of the in vitro plants have a length of 4-6 mm
  • PAT gene phsphinothricin acetyltransferase gene
  • the plants are planted in a mixture of clay, sand, vermiculite and common earth in a ratio of 3: 1: 1: 1 and during the first 3 days after transplanting adapted to the soil culture at 90 - 100% relative humidity
  • the cultivation takes place in a climatic chamber with 14 h light period approx. 25000 lux at plant height at a day /
  • Peptide 1 NH 2 -GTGGLRDTVENC-COOH (Seq. ID No. 3) This peptide was coupled to the KLH carrier ("keyhole limpet homocyanin") and then used to produce polyclonal antibodies in rabbits (Eurogentec, Seraing , Belgium).
  • the resulting antibody was named anti-SSI.
  • the anti-SSI antibody was then used to screen a maize cDNA library for sequences encoding soluble maize starch synthases.
  • a cDNA library from Endosperm-polyA + RNA created in the vector ⁇ -ZAP, was used.
  • To analyze the phage plaques these were transferred to nitrocellulose filters, which had previously been used for 30-60 min. were incubated in a 10 mM IPTG solution and then dried on filter paper. The transfer took place at 37 ° C. for 3 h. The filters were then for 30 min. incubated at room temperature in block reagent and twice for 5-10 min. washed in TBST buffer.
  • the filters were shaken with the polyelonal anti-SSI antibody in a suitable dilution for 1 h at room temperature or for 16 h at 4 ° C. Plaques which expressed a protein which was recognized by the antibody anti-SSI were identified using the "Blotting detection kit for rabbit antibodies RPN 23" (Amersham UK) according to the manufacturer's instructions.
  • Phage clones from the cDNA library expressing a protein recognized by the anti-SSI antibody were further purified using standard procedures. With the aid of the in vivo excision method (Stratagene), E. coli clones were obtained from positive phage clones which contain a double-stranded pBluescript II SK plasmid with the respective cDNA insert between the EcoRI and the Xho I interface of the polylinker. After checking the size and the restriction A suitable clone was subjected to a sequence analysis after the insertions.
  • the plasmid pSSS1 was isolated from an E. coli clone obtained according to Example 1 and its cDNA insertion by standard methods using the dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467) certainly.
  • the insertion is 2383 bp long and represents a partial cDNA.
  • the nucleotide sequence is shown under Seq ID No. 1 specified.
  • the corresponding amino acid sequence is under Seq ID No. 2 shown.
  • the vector pUBIbar (see FIG. 1) was linearized with the restriction enzyme Hpal and dephosphorylated with alkaline phosphatase.
  • the cDNA (approx. 2.4 kb) isolated according to Example 1, which had been obtained as an EcoRV / Smal fragment from the pBluescriptSK plasmid, was cloned into the linearized vector.
  • a plasmid was identified by restriction analysis which contained the cDNA encoding the corn starch synthase in antisense orientation in relation to the promoter. This plasmid was called pUBI-bar-aMasy.
  • This vector contains an ubiquitin promoter and an intron from maize (Christensen et al., Plant Mol. Biol. 18 (1992), 675-689), the transcription termination signal of the nopaline synthase gene from A. tumefaciens (Depicker et al., J Mol. Appl. Genet. 1 (1982), 561-573), the bar marker gene (Thompson et al., EMBO J.
  • the plasmid between the intron and the nos terminator in anti-sense orientation to the ubiquitin promoter contains the cDNA encoding the corn starch synthase.
  • the plasmid is shown in Figure 2.
  • the vector pUBI-bar-aMasy was introduced into maize protoplasts using the method described above. 4.8 ⁇ 10 7 protoplasts and 100 ⁇ g plasmid DNA were used.
  • GGT TCC ATC GAT AAC ACA GTA GTT GTG GCA AGT GAG CAA GAT TCT GAG 382
  • GGT CTC AAT CAG CTA TAT GCT ATG CAG TAT GGC ACA GTT CCT GTT GTC 1678 Gly Leu Asn Gin Leu Tyr Ala Met Gin Tyr Gly Thr Val Pro Val Val 545 550 555

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Abstract

L'invention concerne des molécules d'acide nucléique codant des enzymes intervenant dans la synthèse d'amidon dans des plantes. Ces enzymes constituent une nouvelle isoforme de la synthase d'amidon soluble obtenue à partir de maïs. L'invention concerne en outre des vecteurs contenant de telles molécules d'acide nucléique, ainsi que des cellules hôtes transformées avec les molécules d'acide nucléique décrites, notamment des cellules végétales transformées ainsi que des plantes pouvant être régénérées par ces dernières et présentant une activité accrue ou réduite des protéines décrites.
EP97923925A 1996-05-17 1997-05-16 Molecules d'acide nucleique codant des synthases d'amidon solubles obtenues a partir de mais Withdrawn EP0904389A1 (fr)

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DE19619918A DE19619918A1 (de) 1996-05-17 1996-05-17 Nucleinsäuremoleküle codierend lösliche Stärkesynthasen aus Mais
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PCT/EP1997/002527 WO1997044472A1 (fr) 1996-05-17 1997-05-16 Molecules d'acide nucleique codant des synthases d'amidon solubles obtenues a partir de mais

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CA2255538A1 (fr) 1997-11-27
DE19619918A1 (de) 1997-11-20
JP2000511049A (ja) 2000-08-29
WO1997044472A1 (fr) 1997-11-27
US20020088023A1 (en) 2002-07-04
AU2956997A (en) 1997-12-09
US6307124B1 (en) 2001-10-23
US6635804B2 (en) 2003-10-21
AU725197B2 (en) 2000-10-05

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