Molecular and General Genetics MGG

, Volume 225, Issue 2, pp 289–296 | Cite as

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

  • R. G. F. Visser
  • I. Somhorst
  • G. J. Kuipers
  • N. J. Ruys
  • W. J. Feenstra
  • E. Jacobsen


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.

Key words

Amylose content Antisense RNA Dominant (hemizygous) suppression Granule-bound starch synthase Transgenic potato 


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  1. 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 321:446–449Google Scholar
  2. Benfey PN, Chua N-H (1989) Regulated genes in transgenic plants. Science 244:174–181Google Scholar
  3. 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 8:2195–2202Google Scholar
  4. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  5. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in E. coli. J Mol Biol 138:174–207Google Scholar
  6. 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 17:833–843Google Scholar
  7. Crowley TE, Nellen W, Gomer RH, Firtel R (1985) Phenocopy of discoidin I-minus mutants by antisense transformation in Dictyostelium. Cell 43:633–641Google Scholar
  8. Delauney AJ, Tabaeizadeh Z, Verma DPS (1988) A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proc Natl Acad Sci USA 85:4300–4304Google Scholar
  9. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Reports 1:19–21Google Scholar
  10. Ecker JR, Davis RW (1986) Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83:5372–5376Google Scholar
  11. Green PJ, Pines O, Inouye M (1986) The role of antisense RNA in gene regulation. Annu Rev Biochem 55:569–597Google Scholar
  12. Hergersberg M (1988) Molekulare Analyse des waxy Gens aus Solanum tuberosum und Expression von waxy antisense RNA in transgenen Kartoffeln. Inaugural Dissertation, KölnGoogle Scholar
  13. 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 83:4794–4798Google Scholar
  14. 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 75:217–221Google Scholar
  15. 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 31:241–246Google Scholar
  16. Izant JG, Weintraub HW (1984) Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysis. Cell 36:1007–1015Google Scholar
  17. Izant JG, Weintraub HW (1985) Constitutive and conditional expression of exogenous and endogenous genes by anti-sense RNA. Science 229:345–352Google Scholar
  18. 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 44:43–48Google Scholar
  19. Kim SK, Wold BJ (1985) Stable reduction of thymidine kinase activity in cells expressing high levels of antisense RNA. Cell 42:129–138Google Scholar
  20. 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 333:866–869Google Scholar
  21. van der Krol AR, Mol JNM, Stuitje AE (1989) Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6:958–976Google Scholar
  22. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  23. McGarry TJ, Lindquist S (1986) Inhibition of heat shock protein synthesis by heat-inducible antisense RNA. Proc Natl Acad Sci USA 83:399–403Google Scholar
  24. 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 81:1966–1970Google Scholar
  25. Mol JNM, Stuitje AR, van der Krol AR (1989) Genetic manipulation of floral pigmentation genes. Plant Mol Biol 13:287–294Google Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  27. 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 83:6935–6939Google Scholar
  28. 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 l3:399–409Google Scholar
  29. 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 84:8439–8443Google Scholar
  30. 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 11:301–310Google Scholar
  31. 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 13:303–312Google Scholar
  32. Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85:8805–8809Google Scholar
  33. 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 334:724–726Google Scholar
  34. Tautz D, Renz M (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132:14–19Google Scholar
  35. 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 78:1421–1425Google Scholar
  36. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar
  37. Visser RGF (1989) Manipulation of the starch composition of Solanum tuberosum L. using Agrobacterium rhizogenes mediated transformation. PhD Thesis, University of Groningen, pp 97–117Google Scholar
  38. 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 78:594–600Google Scholar
  39. 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 78:705–714Google Scholar
  40. 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 12:329–337Google Scholar
  41. 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 64:185–192Google Scholar
  42. Visser RGF, Feenstra WJ, Jacobsen E (1990) In: Mol JNM, van der Krol AR (eds) Applications of antisense nucleic acids. Marcel Dekker, NY, in pressGoogle Scholar
  43. Vos-Scheperkeuter GH, de Boer W, Visser RGF, Feenstra WJ, Witholt B (1986) Identification of granule-bound starch synthase in potato tubers. Plant Physiol 82:411–416Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • R. G. F. Visser
    • 1
  • I. Somhorst
    • 2
  • G. J. Kuipers
    • 1
  • N. J. Ruys
    • 1
  • W. J. Feenstra
    • 2
  • E. Jacobsen
    • 1
  1. 1.Department of Plant Breeding (IvP)Agricultural UniversityWageningenThe Netherlands
  2. 2.Department of GeneticsUniversity of GroningenHarenThe Netherlands

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