Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1999-11-09
2003-02-18
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S285000, C800S286000, C800S298000, C800S320200, C800S320000, C800S320100, C800S320300, C800S317200, C800S317400, C800S306000, C800S312000, C800S322000, C435S320100, C435S252300, C435S419000, C435S430000, C435S468000, C435S101000, C435S069100, C536S023600, C536S023200
Reexamination Certificate
active
06521816
ABSTRACT:
The present invention relates to nucleic acid molecules encoding an R1-protein from rice as well as to methods and recombinant DNA molecules for the production of transgenic plant cells and plants synthesizing modified starch. The invention also relates to the transgenic plant cells and plants resulting from these methods and to the starch obtainable from the transgenic plant cells and plants.
The polysaccharide starch, which constitutes one of the most important storage substances in plants, is not only used in the area of foodstuffs but also plays a significant role as a regenerative material in the manufacturing of industrial products. In order to enable the use of this raw material in as many areas as possible, it is necessary to obtain a large variety of substances as well as to adapt these substances to the varying demands of the processing industry.
Although starch consists of a chemically homogeneous basic component, namely glucose, it does not constitute a homogeneous raw material. It is rather a complex mixture of various types of molecules which differ from each other in their degree of polymerization and in the degree of branching of the glucose chains. One differentiates particularly between amylose-starch, a basically non-branched polymer made up of &agr;-1,4-glycosidically branched glucose molecules, and amylopectin-starch which in turn is a mixture of more or less heavily branched glucose chains. The branching results from the occurrence of &agr;-1,6-glycosidic interlinkings.
The molecular structure of starch which is mainly determined by its degree of branching, the amylose/amylopectin ration, the average chain-length and the occurrence of phosphate groups is significant for important functional properties of starch or, respectively, its watery solutions. Important functional properties are for example solubility of the starch, tendency to retrogradation, capability of film formation, viscosity, pastification properties, i.e. binding and gluing properties, as well as cold resistance. The starch granule size may also be significant for the various uses. The production of starch with a high amylose content is particularly significant. Furthermore, modified starch contained in plant cells may, under certain conditions, favorably alter the behavior of the plant cell. For example, it would be possible to decrease the starch degradation during the storage of the starch-containing organs such as seeds and tubers prior to their further processing by, for example, starch extraction. Moreover, there is some interest in producing modified starches which would render plant cells and plant organs containing this starch more suitable for further processing, such as for the production of popcorn or corn flakes from maize or of French fries, crisps or potato powder from potatoes. There is a particular interest in improving the starches in such a way, that they show a reduced “cold sweetening”, i.e. a decreased release of reduced sugars (especially glucose) during long-term storage at low temperatures.
Furthermore, in the case of rice, it is known that the change of the starch's physico-chemical properties influences the cooking and eating qualities of rice grains. The possibility of altering and fine-tuning these properties would permit the development of new rice varieties with a specific quality type. Quality types are usually based on the starch properties or textures of cooked rice, specifically apparent amylose content (AC), final starch gelatinization temperature (GT), and gel consistency (GC) of milled rice (Juliano, Cereal Foods World 43 (1998), 207-222).
Starch which can be isolated from plants is often adapted to certain industrial purposes by means of chemical modifications which are usually time-consuming and expensive. Therefore it is desirable to find possibilities to produce plants synthesizing a starch the properties of which already meet the demands of the processing industry. Conventional methods for producing such plants are classical breeding methods and the production of mutants both of which are, however, expensive and time consuming. Alternatively, plants synthesizing starch with altered properties may be produced by means of recombinant DNA techniques. However, in order to make use of recombinant DNA techniques, DNA sequences are required the gene products of which influence starch synthesis, starch modification or starch degradation, in particular sequences of such an important starch-synthesizing plant as rice.
Therefore, the problem underlying the present invention is to provide nucleic acid molecules and methods which allow for the alteration of plants in such a way, that they synthesize a starch which differs from starch naturally synthesized in plants with respect to its physical and/or chemical properties (these properties in turn influence, for example, the cooking properties and/or the nutritional value of the harvestable parts of these plants) and which starch is therefore more suitable for general and/or particular uses.
This problem is solved by the provision of the embodiments described in the claims.
Therefore, the present invention relates to nucleic acid molecules encoding a protein, in particular from rice, comprising the amino acid sequence indicated in Seq. ID No. 2. Such proteins are present in the plastids of plant cells, particularly in the plastids of cells from rice. In the scope of the present invention the protein encoded by the described nucleic acid molecules is referred to as an R1-protein. It is suspected that this protein exists in the plastids in a form bound to the starch granules as well as in a soluble form. Furthermore, this protein is involved in the phosphorylation of starch.
The present invention further relates to nucleic acid molecules comprising the nucleotide sequence indicated in Seq. ID No. 1, particularly the coding region indicated in Seq. ID No. 1.
The present invention also relates to nucleic acid molecules encoding a polypeptide comprising the amino acid sequence as encoded by the cDNA insert of plasmid DSM 12439.
Furthermore, the present invention relates to nucleic acid molecules comprising the coding region contained in the cDNA insert of plasmid DSM 12439.
Nucleic acid molecules encoding a protein in particular from rice, which occurs in the plastids of the cells, and hybridizing to the above-mentioned nucleic acid molecules of the invention or their complementary strand are also the subject matter of the present invention. In this context the term “hybridization” signifies hybridization under conventional hybridizing conditions, preferably under stringent conditions as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2
nd
Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
More preferably hybridization occurs under the following conditions:
Hybridization
2 × SSC; 10 × Denbard's solution (Fikoll 400 + PEG
buffer:
+ BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM
Na
2
HPO
4
; 250 &mgr;g/ml herring sperm DNA;
50 &mgr;g/ml tRNA; or 0.25M sodiumphospbate buffer
pH 7.2
1 mM EDTA
7% SDS
Hybridization
T = 65 to 68° C.
temperature
Washing
0.2 × SSC; 0.1% SDS
buffer:
Washing
T = 65 to 68° C.
temperature
Nucleic acid molecules hybridizing to the molecules according to the invention may be isolated e.g. from genomic or from cDNA libraries produced in particular from rice cells or tissue.
The identification and isolation of such nucleic acid molecules may take place by using the molecules according to the invention or parts of these molecules or, as the case may be, the reverse complementary strands of these molecules, e.g. by hybridization according to standard methods (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
As a probe for hybridization e.g. nucleic acid molecules may be used which exactly or basically contain the nucleotide sequence indicated under Seq. ID No. 1 or parts thereof The DNA fra
Fox David T.
Kalinowski Grant
Kubelik Anne
PlantTec Biotechnologie GmbH Forschung und Entwicklung
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