Summary
Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.
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Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD (1986) Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature 321:446–449
Benfey PN, Chua N-H (1989) Regulated genes in transgenic plants. Science 244:174–181
Benfey PN, Ren L, Chua N-H (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195–2202
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in E. coli. J Mol Biol 138:174–207
Cornelissen M, Van de Wiele M (1989) Both RNA level and translation efficiency are reduced by anti-sense RNA in transgenic tobacco. Nucleic Acids Res 17:833–843
Crowley TE, Nellen W, Gomer RH, Firtel R (1985) Phenocopy of discoidin I-minus mutants by antisense transformation in Dictyostelium. Cell 43:633–641
Delauney AJ, Tabaeizadeh Z, Verma DPS (1988) A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proc Natl Acad Sci USA 85:4300–4304
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Reports 1:19–21
Ecker JR, Davis RW (1986) Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83:5372–5376
Green PJ, Pines O, Inouye M (1986) The role of antisense RNA in gene regulation. Annu Rev Biochem 55:569–597
Hergersberg M (1988) Molekulare Analyse des waxy Gens aus Solanum tuberosum und Expression von waxy antisense RNA in transgenen Kartoffeln. Inaugural Dissertation, Köln
Holt JT, Gopal TV, Moulton AD, Nienhuis AW (1986) Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation. Proc Natl Acad Sci USA 83:4794–4798
Hovenkamp-Hermelink JHM, Jacobsen E, Ponstein AS, Visser RGF, Vos-Scheperkeuter GH, Bijmolt EW, de Vries JN, Witholt B, Feenstra WJ (1987) Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor Appl Genet 75:217–221
Hovenkamp-Hermelink JHM, de Vries JN, Adamse P, Jacobsen E, Witholt B, Feenstra WJ (1988) Rapid estimation of the amylose/amylopectin ratio in small amounts of tuber and leaf tissue of the potato. Potato Res 31:241–246
Izant JG, Weintraub HW (1984) Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysis. Cell 36:1007–1015
Izant JG, Weintraub HW (1985) Constitutive and conditional expression of exogenous and endogenous genes by anti-sense RNA. Science 229:345–352
Jacobsen E, Hovenkamp-Hermelink JHM, Krijgsheld HT, Nijdam H, Pijnacker LP, Wiltholt B, Feenstra WJ (1989) Phenotypic and genotypic characterization of an amylose-free starch mutant of the potato. Euphytica 44:43–48
Kim SK, Wold BJ (1985) Stable reduction of thymidine kinase activity in cells expressing high levels of antisense RNA. Cell 42:129–138
van der Krol AR, Lenting PE, Veenstra J, van der Meer IM, Kees RE, Gerats AGM, Mol JNM, Stuitje AR (1988) Expression of an antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333:866–869
van der Krol AR, Mol JNM, Stuitje AE (1989) Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6:958–976
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
McGarry TJ, Lindquist S (1986) Inhibition of heat shock protein synthesis by heat-inducible antisense RNA. Proc Natl Acad Sci USA 83:399–403
Mizuno T, Chou M, Inouye M (1984) A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci USA 81:1966–1970
Mol JNM, Stuitje AR, van der Krol AR (1989) Genetic manipulation of floral pigmentation genes. Plant Mol Biol 13:287–294
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Offringa IA, Melchers LS, Regensburg-Tuink AJG, Costantino P, Schilperoort RA, Hooykaas PJJ (1986) Complementation of Agrobacterium tumefaciens tumor inducing aux mutants by genes from the Tr-region of the Ri-plasmid of Agrobacterium rhizogenes. Proc Natl Acad Sci USA 83:6935–6939
Robert LS, Donaldson PA, Ladaique C, Altosaar I, Arnison PG, Fabijanski SF (1989) Antisense RNA inhibition of β-glucuronidase gene expression in transgenic tobacco plants. Plant Mol Biol l3:399–409
Rothstein SJ, DiMaio J, Strand M, Rice D (1987) Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proc Natl Acad Sci USA 84:8439–8443
Sandler SJ, Stayton M, Townsend JA, Ralston ML, Bedbrook JR, Dunsmuir P (1988) Inhibition of gene expression in transformed plants by antisense RNA. Plant Mol Biol 11:301–310
Schuch W, Bird CR, Ray J, Smith CIS, Watson CF, Morris PC, Gray JE, Arnold C, Seyman GB, Tucker GA, Grierson D (1989) Control and manipulation of gene expression during tomato fruit ripening. Plant Mol Biol 13:303–312
Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85:8805–8809
Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724–726
Tautz D, Renz M (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132:14–19
Tomizawa JI, Itoh T, Selzer G, Som T (1981) Inhibition of ColEI primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci USA 78:1421–1425
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Visser RGF (1989) Manipulation of the starch composition of Solanum tuberosum L. using Agrobacterium rhizogenes mediated transformation. PhD Thesis, University of Groningen, pp 97–117
Visser RGF, Jacobsen E, Witholt B, Feenstra WJ (1989a) Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes. Theor Appl Genet 78:594–600
Visser RGF, Hesseling-Meinders A, Jacobsen E, Nijdam H, Witholt B, Feenstra WJ (1989b) Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.) Theor Appl Genet 78:705–714
Visser RGF, Jacobsen E, Hesseling-Meinders A, Schans MJ, Witholt B, Feenstra WJ (1989c) Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments. Plant Mol Biol 12:329–337
Visser RGF, Hergersberg M, van der Leij FR, Jacobsen E, Witholt B, Feenstra WJ (1989d) Molecular cloning and partial characterization of the gene for granule-bound starch synthase from a wild type and an amylose-free potato (Solanum tuberosum L.). Plant Sci 64:185–192
Visser RGF, Feenstra WJ, Jacobsen E (1990) In: Mol JNM, van der Krol AR (eds) Applications of antisense nucleic acids. Marcel Dekker, NY, in press
Vos-Scheperkeuter GH, de Boer W, Visser RGF, Feenstra WJ, Witholt B (1986) Identification of granule-bound starch synthase in potato tubers. Plant Physiol 82:411–416
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Visser, R.G.F., Somhorst, I., Kuipers, G.J. et al. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Molec. Gen. Genet. 225, 289–296 (1991). https://doi.org/10.1007/BF00269861
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DOI: https://doi.org/10.1007/BF00269861