Skip to main content
SpringerLink
Log in

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS ‘gene’ dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially ‘down mutations’ in essential genes where only a short 5′ sequence of the mRNA is required. They also suggest that the ‘position effect’ on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York (1987).

    Google Scholar 

  2. Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).

    PubMed  Google Scholar 

  3. Boyer HW, Roulland-Dussoix D: A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41: 459–472 (1969).

    Article  PubMed  Google Scholar 

  4. Buchanan-Wollaston V, Passiatore JE, Cannon F: The mob and oriT functions of a bacterial plasmid promote its transfer to plants. Nature 328: 172–175 (1987).

    Article  Google Scholar 

  5. Colman J, Hirashima A, Inokuchi Y, Green PJ, Inouye M: A novel immune system against bacteriophage infection using complementary RNA (micRNA). Nature 315: 601–603 (1985).

    PubMed  Google Scholar 

  6. Crowley TE, Nellen W, Gomer RH, Firtel RA: Phenotype of discoidin 1-minus mutants by antisense transformation in Dictyostelium. Cell 43: 33–641 (1985).

    Article  Google Scholar 

  7. Delauney AJ, Tabaeizadeh Z, Verma DPS: A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proc Natl Acad Sci USA 85: 4300–4304 (1988).

    Google Scholar 

  8. Dhaese P, De Greve H, Gielen J, Seurinck J, Montaguvan M, Schell J: Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO J 2: 419–426 (1983).

    PubMed  Google Scholar 

  9. Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351 (1980).

    PubMed  Google Scholar 

  10. Dunsmuir P, Bond D, Lee K, Gidoni D, Townsend J: in Gelvin SB, Schilperoort RA, Verma DPS (eds), Plant Molecular Biology Manual C1: 1–17. Kluwer Academic Publishers, Dordrecht (1988).

    Google Scholar 

  11. Ecker JR, Davis RW: Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83: 5372–5376 (1986).

    Google Scholar 

  12. Elzenvan der PJM, Townsend J, Lee KY, Bedbrook JR: A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5: 299–302 (1985).

    Google Scholar 

  13. Green PJ, Pines O, Inouye M: The role of antisense RNA in gene regulation. Ann Rev Biochem 55: 569–597 (1986).

    PubMed  Google Scholar 

  14. Hernandez G, Cannon F, Cannon M: The effect of presumptive polyadenylation signals on the expression of the CAT gene in transgenic tobacco. Plant Cell Reports 8: 195–198 (1989).

    Google Scholar 

  15. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1983).

    Google Scholar 

  16. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  17. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    PubMed  Google Scholar 

  18. Jefferson RA: Assaying chimeric genes in plants: The GUS Gene Fusion System. Plant Mol Biol Rep 5: 387–405 (1987).

    Google Scholar 

  19. Kim SK, Wold BJ: Stable reduction of thymidine kinase activity in cells expressing high levels of antisense RNA. Cell 42: 129–138 (1985).

    PubMed  Google Scholar 

  20. Krolvan der AR, Lenting PE, Veenstra J, Meervan der IM, Koes RE, Gerats AGM, Mol JNM, Stuitje AR: An antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333: 866–869 (1988).

    Article  Google Scholar 

  21. Krolvan der AR, Mol JNM, Stuitje AR: Modulation of eucaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6: 958–974 (1988).

    PubMed  Google Scholar 

  22. Leutwiler LS, Meyerowitz EM, Tobin EM: Structure and expression of three light-harvesting chlorophyll α/b-binding protein genes in Arabidopsis thaliana. Nucl Acids Res 14: 4051–4064 (1986).

    PubMed  Google Scholar 

  23. Marsh JL, Erfle M, Wykes EJ: The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional activation. Gene 32: 481–485 (1984).

    Article  PubMed  Google Scholar 

  24. Melton D: Injected antisense RNA's specifically block messenger RNA translation in vivo. Proc Natl Acad Sci USA 82: 144–148 (1985).

    PubMed  Google Scholar 

  25. Mizuono T, Chou M, Inouye M: A unique mechanism regulating gene expression: translation inhibition by complementary RNA transcript (micRNA). Proc Natl Acad Sci USA 81: 1966–1970 (1984).

    PubMed  Google Scholar 

  26. Odell JT, Nagy F, Chua N-H: Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812 (1985).

    PubMed  Google Scholar 

  27. Rothstein SJ, DiMaio J, Strand M, Rice D: Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proc Natl Acad Sci USA 84: 8439–8443 (1987).

    Google Scholar 

  28. Sandler SJ, Stayton M, Townsend JA, Ralston ML, Bedbrook JR, Dunsmuir P: Inhibition of gene expression in transformed plants by antisense RNA. Plant Mol Biol 11: 301–310 (1988).

    Google Scholar 

  29. Sheehy RE, Kramer M, Hiatt WR: Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85: 8805–8809 (1988).

    Google Scholar 

  30. Simons RW, Kleckner N: Translational control of IS10 transposition. Cell 34: 683–691 (1983).

    PubMed  Google Scholar 

  31. Smith CJS, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D: Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334: 724–726 (1988).

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Bio Technica International, Inc., 85 Bolton Street, 02140, Cambridge, MA, USA

    Maura Cannon & Jerry Platz

  2. Plant Pathology Dept., University of Massachusetts, 01003, Amherst, MA, USA

    Maureen O'Leary

  3. Dept. of Molecular Biology, Massachusetts General Hospital, 02114, Boston, MA, USA

    Frank Cannon

Authors
  1. Maura Cannon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jerry Platz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maureen O'Leary
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cathleen Sookdeo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frank Cannon
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cannon, M., Platz, J., O'Leary, M. et al. Organ-specific modulation of gene expression in transgenic plants using antisene RNA. Plant Mol Biol 15, 39–47 (1990). https://doi.org/10.1007/BF00017722

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00017722

Key words

Search

Navigation