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
1998-11-05
2001-07-03
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
C800S317200, C800S320000, C800S320100, C435S069100, C435S101000, C435S252300, C435S320100, C435S412000, C435S417000, C435S419000, C536S023600
Reexamination Certificate
active
06255563
ABSTRACT:
FIELD OF THE INVENTION
Nucleic acid molecules coding for debranching enzymes from potato The present invention relates to nucleic acid molecules encoding proteins from potato with the enzymatic activity of a debranching enzyme. The invention further relates to transgenic plants and plant cells, in which an amylopectin with an altered degree of branching is synthesized due to the expression of an additional debranching enzyme activity from potato or due to the inhibition of an endogeneous debranching enzyme activity. The invention also relates to the starch obtainable from said transgenic plant cells and plants.
Starch plays an important role as storage substance in a multitude of plants and also as a regenerative, industrially usable raw material and has gained increasing significance. For the industrial use of starch it is necessary that it meets the demands of the processing industry with respect to its structure, form and/or other physico-chemical parameters. In order to enable the use in as many areas as possible it is furthermore necessary to achieve a large variety of substances. The polysaccharide starch is made up of chemically homogeneous basic components, namely the glucose molecules. However, it constitutes a highly complex mixture of various types of molecules which differ from each other in their degree of polymerization and in the degree of branching. One differentiates between amylose-starch, a basically non-branched polymer made up of &agr;-1,4-glycosidically branched glucose molecules, and amylopectin-starch, a branched polymer, in which the branching results from additional &agr;-1,6-glycosidic interlinkings.
In plants used typically for the production of starch, such as maize or potato, the synthesized starch consists of approximately 25% amylose-starch and of about 75% amylopectin-starch. In the case of maize, for example, a further branched polysaccharide, apart from amylopectin, occurs, namely the so-called phytoglycogen which differs from amylopectin by exhibiting a higher degree of branching and different solubility (see e.g. Lee et al., Arch. Biochem. Biophys. 143 (1971), 365-374; Pan and Nelson, Plant Physiol. 74 (1984), 324-328). In the scope of the present application the term amylopectin is used in such a way as to comprise the phytoglycogen.
With respect to the homogeneity of the basic component starch for its use in the industrial area, starch-producing plants are needed which contain, for example, only the component amylopectin or only the component amylose. For a number of other uses plants are needed that synthesize amylopectin types with different degrees of branchings.
Such plants may for example be obtained by breeding or by means of mutagenesis techniques. It is known for various plant species, such as for maize, that by means of mutagenesis varieties may be produced in which only amylopectin is formed. Also in the case of potato a genotype was produced from a haploid line by means of chemical mutagenesis. Said genotype does not form amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221).
Apart from conventional breeding and mutagenesis techniques, recombinant DNA techniques are now increasingly used in order to specifically interfere with the starch metabolism of starch storing plants. A prerequisite for this is that DNA sequences be provided which encode enzymes involved in the starch metabolism. In the case of potato, for example, DNA sequences have by now been found which encode a granule-bound starch synthase or a branching enzyme (Q enzyme), and they have been used in order to genetically modify plants.
For a further targeted modification of the starch in plants, in particular of the degree of branching of starch synthesized in plants by means of recombinant DNA techniques, it is still necessary to identify DNA sequences that encode enzymes participating in the starch metabolism, particularly in the branching of starch molecules.
Apart from the Q enzymes that introduce branchings into starch molecules, enzymes occur in plants which are capable of dissolving branchings. These enzymes are called debranching enzymes.
In the case of sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) could only prove the occurrence of one debranching enzyme, apart from five endo- and two exoamylases. This enzyme having a size of approximately 100 kD and an optimum pH value of 5.5 is located within the chloroplasts. A debranching enzyme was also described for spinach. The debranching enzyme from spinach as well as that from sugar beet exhibit a fivefold lower activity in a reaction with amylopectin as substrate when compared to a reaction with pullulan as a substrate (Ludwig et al., Plant Physiol. 74 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284). The isolation of a cDNA encoding a debranching enzyme was described for spinach (Renz et al., Plant Physiol. 108 (1995), 1342).
The existence of a debranching enzyme for maize has been described in the prior art. The corresponding mutant was designated su (sugary). The gene of the sugary locus was cloned recently (see James et al., Plant Cell 7 (1995), 417-429). In the case of the agriculturally significant starch-storing cultured plant potato, the activity of a debranching enzyme was examined by Hobson et al. (J. Chem. Soc., (1951), 1451). It was proven that the respective enzyme, contrary to the Q enzyme, does not exhibit any activities leading to an elongation of the polysaccharide chain, but merely hydrolyses &agr;-1,6-glycosidic bonds. Methods for the purification of a debranching enzyme from potato as well as partial peptide sequences of the purified protein have already been described (WO 95/04826).
So far no indication as to the existence of further debranching enzyme types from potato could be found. Should this, however, be the case, all debranching enzyme types occurring in potato would have to be identified and the corresponding genes or cDNA sequences would have to be isolated in order to produce transgenic potato plants that do no longer exhibit any debranching enzyme activity for the purpose of achieving a modification of the degree of branching of the amylopectin starch.
Therefore, the technical problem underlying the present invention is to identify further debranching enzymes possibly occurring in potato and to isolate corresponding nucleic acid molecules encoding these enzymes.
This problem is solved by the provision of the embodiments as defined in the claims.
SUMMARY OF THE INVENTION
Thus, the present invention relates to nucleic acid molecules encoding proteins with the biological activity of a debranching enzyme from potato. Such a nucleic acid molecule preferably encodes a protein with the biological activity of a debranching enzyme from potato that exhibits the amino acid sequence depicted in SEQ ID No. 2. In a particularly preferred embodiment such a nucleic acid molecule comprises the nucleotide sequence depicted under SEQ ID No. 1, in particular the coding region.
The present invention also relates to nucleic acid molecules encoding proteins with the biological activity of a debranching enzyme from potato and hybridizing to one of the above-described nucleic acid molecules or to the complementary strand thereof. Furthermore, the present invention relates to nucleic acid molecules the sequence of which differs from the sequences of the above-mentioned nucleic acid molecules due to a degeneracy of the genetic code, and which encode a protein exhibiting the biological activity of a debranching enzyme from potato.
The term “from potato” means that the debranching enzymes encoded by the nucleic acid molecules of the invention are typical for the species Solanum tuberosum, i.e. they either occur naturally in such plants, for example encoded by genomic or RNA molecules or by molecules derived therefrom. Derived molecules may for example be produced by the reverse transcription of RNA molecules, amplification, mutation, deletion, substitution, insertion etc. I.e. the term also comprises enzymes encoded by alleles and derivatives of sequences naturally
Emmermann Michael
Kossmann Jens
Fish & Neave
Fox David T.
Haley Jr. James F.
Planttec Biotechnologie GmbH
Su Li
LandOfFree
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