Genetically engineered modification of potato to form...

Details Genetically engineered modification of potato to form... Genetically engineered modification of potato to form...

1993-11-24

2004-08-31

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, C800S286000, C800S287000, C800S317200, C435S101000, C435S320100, C435S417000, C435S419000, C536S023600, C536S024100, C536S024500

Reexamination Certificate

active

06784338

ABSTRACT:

BACKGROUND OF THE INVENTION
Starch in various forms is of great import in the food and paper industry. In future, starch will also be a great potential for producing polymers which are degradble in nature, e.g. for use as packing material. Many different starch products are known which are produced by derivatisation of native starch originating from, inter alia, maize and potato. Starch from potato and maize, respectively, is competing in most market areas.
In the potato tuber, starch is the greatest part of the solid matter. About ¼ to ⅕ of the starch in potato is amylose, while the remainder of the starch is amylopectin. These two components of the starch have different fields of application, and therefore the possibility of producing either pure amylose or pure amylopectin is most interesting. The two starch components can be produced from common starch, which requires a number of process steps and, consequently, is expensive and complicated.
It has now proved that by genetic engineering it is possible to modify potato so that the tubers merely produce mainly starch of one or the other type. As a result, a starch quality is obtained which can compete in the areas where potato starch is normally not used today. Starch from such potato which is modified in a genetically engineered manner has great potential as a food additive, since it has not been subjected to any chemical modification process.
Starch Synthesis
The synthesis of starch and the regulation thereof are presently being studied with great interest, both on the level of basic research and for industrial application. Although much is known about the assistance of certain enzymes in the transformation of saccharose into starch, the biosynthesis of starch has not yet been elucidated. By making researches above all into maize, it has, however, been possible to elucidate part of the ways of synthesis and the enzymes participating in these reactions. The most important starch-synthesising enzymes for producing the starch granules are the starch synthase and the branching enzyme. In maize, three forms of starch synthase have so far been demonstrated and studied, two of which are soluble and one is insolubly associated with the starch granules. Also the branching enzyme consists of three forms which are probably coded by three different genes (Mac Donald & Preiss, 1985; Preiss, 1988).
The Waxy Gene in Maize
The synthesis of the starch component amylose essentially occurs by the action of the starch synthase alpha-1,4-D-glucane-4-alpha-glucosyl transferase (EC 2.4.1.21) which is associated with the starch granules in the growth cell. The gene coding for this granule-bound enzyme is called “waxy” (=
wx
+
), while the enzyme is called “GBSS” (granule-bound starch synthase).
Waxy locus in maize has been thoroughly characterised both genetically and biochemically. The waxy gene on chromosome
9
controls the production of amylose in endosperm, pollen and the embryo sac. The starch formed in endosperm in normal maize with the wx
+
allele consists to 25% of amylose and to 75% of amylopectin. A mutant form of maize has been found in which the endosperm contains a mutation located to the wx
+
gene, and therefore no functioning GBSS is synthesised. Endosperm from this mutant maize therefore contains merely amylopectin as the starch component. This so-called waxy mutant thus contains neither GBSS nor amylose (Echt & Schwartz, 1981).
The GBSS protein is coded by the wx
+
gene in the cell nucleus but is transported to and active in the amyloplast. The preprotein therefore consists of two components, viz. a 7 kD transit peptide which transfers the protein across the amyloplast membrane, and the actual protein which is 58 kD. The coding region of the wx
+
gene in maize is 3.7 kb long and comprises 14 exons and 13 introns. A number of the regulation signals in the promoter region are known, and two different polyadenylating sequences have been described (Klösgen et al, 1986; Schwartz-Sommer et al, 1984; Shure et al, 1983).
Amylose Enzyme in Potato
In potato, a 60 kD protein has been identified, which constitutes the main granule-bound protein. Since antibodies against this potato enzyme cross-react with GBSS from maize, it is assumed that it is the granule-bound synthase (Vos-Scheperkeuter et al, 1986)., The gene for potato GBSS has, however, so far not been characterised to the same extent as the waxy gene in maize, either in respect of locating or structure.
Naturally occurring waxy mutants have been described for barley, rice and sorghum besides maize. In potato no natural mutant has been found, but a mutant has been produced by X-radiation of leaves from a monohaploid (n=
12
) plant (Visser et al, 1987). Starch isolated from tubers of this mutant contains neither the GBSS protein nor amylose. The mutant is conditioned by a simple recessive gene and is called amf. It may be compared to waxy mutants of other plant species since both the GBSS protein and amylose are lacking. The stability of the chromosome number, however, is weakened since this is quadrupled to the natural number (n=
48
), which can give negative effects on the potato plants (Jacobsen et al, 1990).
Inhibition of Amylose Production
The synthesis of amylose can be drastically reduced by inhibition of the granule-bound starch synthase, GBSS, which catalyses the formation of amylose. This inhibition results in the starch mainly being amylopectin.
Inhibition of the formation of enzyme can be accomplished in several ways, e.g. by:
mutagen treatment which results in a modification of the gene sequence coding for the formation of the enzyme
incorporation of a transposon in the gene sequence coding for the enzyme
genetically engineered modification so that the gene coding for the enzyme is not expressed, e.g. antisense gene inhibition.
FIG. 1
illustrates a specific suppression of normal gene expression in that a complementary antisense nucleotide tide is allowed to hybridise with mRNA for a target gene.
The antisense nucleotide thus is antisense RNA which is transcribed in vivo from a “reversed” gene sequence (Izant, 1989).
By using the antisense technique, various gene functions in plants have been inhibited. The antisense construct for chalcone synthase, polygalacturonase and phosphinotricin acetyltransferase has been used to inhibit the corresponding enzyme in the plant species petunia, tomato and tobacco.
Inhibition of Amylose in Potato
In potato, experiments have previously been made to inhibit the synthesis of the granule-bound starch synthase (GBSS protein) with an antisense construct corresponding to the gene coding for GBSS (this gene is hereinafter called the “GBSS gene” ). Hergersberger (1988) describes a method by which a CDNA clone for the GBSS gene in potato has been isolated by means of a cDNA clone for the wx
+
gene in maize. An antisense construct based on the entire CDNA clone was transferred to leaf discs of potato by means of Agrobacterium tumefaclens. In microtubers induced in vitro from regenerated potato sprouts, a varying and very weak reduction of the amylose content was observed and shown in a diagram. A complete characterisation of the GBSS gene is not provided.
The gene for the GBSS protein in potato has been further characterised in that a genomic wx clone was examined by restriction analysis. However, the DNA sequence of the clone has not been determined (Visser et al, 1989).
Further experiments with an antisense construct corresponding to the GBSS gene in potato have been reported. The antisense construct which is based on a cDNA clone together with the CaMV 35S promoter has been transformed by means of Agrobacterium rhizogenes. According to information, the transformation resulted in a lower amylose content in the potato, but no values have been accounted for (Flavell, 1990).
None of the methods used so far for genetically engineered modification of potato has resulted in potato with practically no amylose-type starch.
The object of the invention therefore is to provide a prac

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