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
2001-01-30
2004-09-21
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
C800S278000, C800S298000, C536S023100, C536S023200, C536S023600, C435S419000, C435S320100
Reexamination Certificate
active
06794558
ABSTRACT:
The present invention relates to nucleic acid molecules which encode a protein with the activity of a potato &agr;-glucosidase, and to processes for the generation of transgenic plant cells and plants which synthesize a modified starch. Moreover, the present invention relates to vectors and host cells comprising the nucleic acid molecules according to the invention, to the plant cells and plants originating from the processes according to the invention, to the starch synthesized by the plant cells and plants according to the invention, and to processes for the production of this starch.
Taking into consideration the increasing importance attached to plant constituents as renewable raw materials, biotechnology research attempts to adapt plant-based raw materials to the needs of the processing industry. To allow renewable raw materials to be used in as many fields of application as possible, it is therefore necessary to provide a multiplicity of substances.
Besides oils, fats and proteins, polysaccharides constitute important renewable raw materials from plants. Besides cellulose, starch, which is one of the most important storage substances in higher plants, occupies a central position amongst the polysaccharides. Besides maize, rice and wheat, potatoes play an important role, in particular in starch production.
The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it is a highly complex mixture of different forms of molecules which differ with regard to their degree of polymerization and the occurrence of branchings of the glucose chains. Starch therefore constitutes no uniform raw material. In particular, we differentiate between amylose starch, an essentially unbranched polymer of &agr;-1,4-glycosidically linked glucose molecules, and amylopectin starch, which, in turn, constitutes a complex mixture of differently branched glucose chains. The branchings are generated by the occurrence of additional &agr;-1,6-glycosidic linkages. In typical plants used for starch production such as, for example, maize or potatoes, the starch synthesized consists of approx. 25% amylose starch and approx. 75% amylopectin starch.
The molecular structure of starch, which is determined to a great extent by the degree of branching, the amylose/amylopectin ratio, the average length and distribution of the side chains, and the presence of phosphate groups, is decisive for important functional properties of starch or its aqueous solutions. Examples of functional properties which must be mentioned in this context are solubility, the retrogradation behavior, the film-forming properties, the viscosity—the color stability, the gelatinization properties, and binding and adhesive properties. The starch granule size may also be of importance for various uses. Also, the generation of high-amylose starches is of particular interest for certain applications. Furthermore, a modified starch present in plant cells can advantageously modify the behavior of the plant cell under certain conditions. For example, it is feasible to reduce starch breakdown during the storage of starch-containing organs, such as, for example, seeds or tubers, prior to their further processing, for example for extracting the starch. It is furthermore of interest to prepared modified starches which lead to plant cells or plant organs containing this starch being better suited to processing, for example in the production of foods such as popcorn or cornflakes from maize, or the production of French fries, chips or potato powder from potatoes. Of particular interest in this context is an improvement of the starches with regard to reduced cold sweetening, i.e. a reduced liberation of reducing sugars (in particular glucose) upon prolonged storage at low temperatures. Potatoes especially are frequently stored at temperatures from 4 to 8° C. in order to minimize starch breakdown during storage. The reducing sugars liberated during this process, in particular glucose, result in undesired browning reactions (so-called Maillard reactions) in the production of French fries or crisps.
The starch which can be isolated from plants is frequently adapted to particular industrial purposes with the aid of chemical modifications which, as a rule, require time and money. It seems therefore desirable to find possibilities of generating plants which synthesize starch whose properties already meet the specific demands of the processing industry and thus combine economical and ecological advantages.
One possibility of providing such plants is, in addition to plant breeding measures, the directed genetic modification of the starch metabolism of starch-producing plants by recombinant methods. However, a prerequisite therefor is the identification and characterization of the enzymes which participate in starch synthesis modificaton and starch breakdown (starch metabolism) and the isolation of the corresponding DNA sequences which encode these enzymes.
The biochemical synthetic pathways which lead to the synthesis of starch are essentially known. In plant cells, starch synthesis takes place in the plastids. In photosynthetically active tissues, these plastids are the chloroplasts, in photosynthetically inactive, starch-storing tissue the amyloplasts.
Important enzymes which are involved in starch metabolism are, for example, the branching enzymes, ADP glucose pyrophosphorylases, granule-bound starch syntheses, soluble starch synthases, debranching enzymes, disproportioning enzymes, plastid starch phosphorylases, the R1 enzymes (R1 proteins), amylases or glucosidases.
It is an object of the present invention to provide other, or alternative, recombinant approaches for modifying the starch metabolism in starch-synthesizing plants (for example rye, barley, oats, maize, wheat, sorghum and millet, sago, rice, peas, marrowfat peas, cassava, potatoes, tomatoes, oilseed rape, soybeans, hemp, flax, sunflowers, cowpeas, mung beans, beans, bananas or arrowroot or) suitable nucleic acid molecules by means of which plant cells can be transformed, thus allowing the synthesis of modified, advantageous starch species.
Such modified starch species exhibit, for example, modifications regarding their degree of branching, the amylose/amylopectin ratio, the phosphate content, the starch granule size and/or the average length and distribution of the side chains (i.e. side chain structure).
It is a further object of the invention to provide methods which allow the generation of transgenic plants which synthesize a modified starch species.
Surprisingly, transgenic plants which have been transformed with the nucleic acid molecules according to the invention synthesize a starch whose physicochemical properties and/or whose side chain structure is modified in the particular manner so that the abovementioned objects are achieved by providing the use forms specified in the claims.
The invention therefore relates to a nucleic acid molecule encoding a protein with the function of a potato &agr;-glucosidase, selected from the group consisting of
a) nucleic acid molecules which encode a protein which encompasses the amino acid sequence stated under SEQ ID NO: 2 or its derivatives or parts,
b) nucleic acid molecules which encompass the nucleotide sequence shown under SEQ ID NO: 1 or its derivatives or parts, or a corresponding ribonucleotide sequence;
c) nucleic acid molecules which hybridize with, or are complementary to, preferably which hybridize specifically with, the nucleic acid molecules stated under a) or b), and
d) nucleic acid molecules whose nucleotide sequence deviates from the sequence of the nucleic acid molecules stated under a), b) or c) owing to the degeneracy of the genetic code.
Accordingly, the present invention relates to a nucleic acid molecule which encodes an &agr;-glucosidase and which comprises an amino acid sequence of SEQ ID NO: 2 or its derivatives or parts in accordance with the cDNA insert of the plasmid (DSM No. 12347). The abovementioned &agr;-glucosidase according to the invention is involved in the starch metabolism of
Aventis CropScience GmbH
Fox David T.
Frommer & Lawrence & Haug LLP
Kallis Russell
LandOfFree
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