Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.).
Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.
Abel
G J
GJ
Institut für Genbiologische Forschung Berlin GmbH, Germany.
Springer
F
F
Willmitzer
L
L
Kossmann
J
J
D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics); D005075:Biological Evolution; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000145:classification / Q000235:genetics*; D030821:Plants, Genetically Modified; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*; D013213:Starch / Q000737:chemistry; D013214:Starch Synthase / Q000145:classification / Q000235:genetics*; D014018:Tissue Distribution
D016375:Antisense Elements (Genetics); D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D013213:Starch; D013214:Starch Synthase