Abstract
A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.
Full Text
The Full Text of this article is available as a PDF (1.9 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Chotigeat W., Watanapokasin Y., Mahler S., Gray P. P. Role of environmental conditions on the expression levels, glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells. Cytotechnology. 1994;15(1-3):217–221. doi: 10.1007/BF00762396. [DOI] [PubMed] [Google Scholar]
- Comai L., Larson-Kelly N., Kiser J., Mau C. J., Pokalsky A. R., Shewmaker C. K., McBride K., Jones A., Stalker D. M. Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide. J Biol Chem. 1988 Oct 15;263(29):15104–15109. [PubMed] [Google Scholar]
- Comai L., Moran P., Maslyar D. Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol. 1990 Sep;15(3):373–381. doi: 10.1007/BF00019155. [DOI] [PubMed] [Google Scholar]
- Jefferson R., Goldsbrough A., Bevan M. Transcriptional regulation of a patatin-1 gene in potato. Plant Mol Biol. 1990 Jun;14(6):995–1006. doi: 10.1007/BF00019396. [DOI] [PubMed] [Google Scholar]
- Klösgen R. B., Weil J. H. Subcellular location and expression level of a chimeric protein consisting of the maize waxy transit peptide and the beta-glucuronidase of Escherichia coli in transgenic potato plants. Mol Gen Genet. 1991 Feb;225(2):297–304. doi: 10.1007/BF00269862. [DOI] [PubMed] [Google Scholar]
- Kumar A., Larsen C. E., Preiss J. Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose:alpha-1,4-glucan, 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J Biol Chem. 1986 Dec 5;261(34):16256–16259. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Müller-Röber B., Sonnewald U., Willmitzer L. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 1992 Apr;11(4):1229–1238. doi: 10.1002/j.1460-2075.1992.tb05167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oakes J. V., Shewmaker C. K., Stalker D. M. Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants. Biotechnology (N Y) 1991 Oct;9(10):982–986. doi: 10.1038/nbt1091-982. [DOI] [PubMed] [Google Scholar]
- Stark D. M., Timmerman K. P., Barry G. F., Preiss J., Kishore G. M. Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase. Science. 1992 Oct 9;258(5080):287–292. doi: 10.1126/science.258.5080.287. [DOI] [PubMed] [Google Scholar]
- Visser R. G., Somhorst I., Kuipers G. J., Ruys N. J., Feenstra W. J., Jacobsen E. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol Gen Genet. 1991 Feb;225(2):289–296. doi: 10.1007/BF00269861. [DOI] [PubMed] [Google Scholar]
- Visser R. G., Somhorst I., Kuipers G. J., Ruys N. J., Feenstra W. J., Jacobsen E. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol Gen Genet. 1991 Feb;225(2):289–296. doi: 10.1007/BF00269861. [DOI] [PubMed] [Google Scholar]
- van der Leij F. R., Visser R. G., Ponstein A. S., Jacobsen E., Feenstra W. J. Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele. Mol Gen Genet. 1991 Aug;228(1-2):240–248. doi: 10.1007/BF00282472. [DOI] [PubMed] [Google Scholar]