Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jul 19;91(15):7301–7305. doi: 10.1073/pnas.91.15.7301

Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase.

K E McBride 1, D J Schaaf 1, M Daley 1, D M Stalker 1
PMCID: PMC44387  PMID: 8041784

Abstract

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.

Full text

PDF
7305

Images in this article

7303Image
on p.7303
7304Image
on p.7304
7305Image
on p.7305

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bendich A. J. Why do chloroplasts and mitochondria contain so many copies of their genome? Bioessays. 1987 Jun;6(6):279–282. doi: 10.1002/bies.950060608. [DOI] [PubMed] [Google Scholar]
  2. Boynton J. E., Gillham N. W., Harris E. H., Hosler J. P., Johnson A. M., Jones A. R., Randolph-Anderson B. L., Robertson D., Klein T. M., Shark K. B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534–1538. doi: 10.1126/science.2897716. [DOI] [PubMed] [Google Scholar]
  3. Carter A. D., Morris C. E., McAllister W. T. Revised transcription map of the late region of bacteriophage T7 DNA. J Virol. 1981 Feb;37(2):636–642. doi: 10.1128/jvi.37.2.636-642.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Danon A., Mayfield S. P. Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins. EMBO J. 1991 Dec;10(13):3993–4001. doi: 10.1002/j.1460-2075.1991.tb04974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deng X. W., Gruissem W. Constitutive transcription and regulation of gene expression in non-photosynthetic plastids of higher plants. EMBO J. 1988 Nov;7(11):3301–3308. doi: 10.1002/j.1460-2075.1988.tb03200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunn J. J., Krippl B., Bernstein K. E., Westphal H., Studier F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene. 1988 Sep 7;68(2):259–266. doi: 10.1016/0378-1119(88)90028-5. [DOI] [PubMed] [Google Scholar]
  9. Hiratsuka J., Shimada H., Whittier R., Ishibashi T., Sakamoto M., Mori M., Kondo C., Honji Y., Sun C. R., Meng B. Y. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet. 1989 Jun;217(2-3):185–194. doi: 10.1007/BF02464880. [DOI] [PubMed] [Google Scholar]
  10. Janssen B. J., Gardner R. C. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol. 1990 Jan;14(1):61–72. doi: 10.1007/BF00015655. [DOI] [PubMed] [Google Scholar]
  11. Jefferson R. A., Kavanagh T. A., Bevan M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987 Dec 20;6(13):3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kay R., Chan A., Daly M., McPherson J. Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes. Science. 1987 Jun 5;236(4806):1299–1302. doi: 10.1126/science.236.4806.1299. [DOI] [PubMed] [Google Scholar]
  13. Kotani H., Ishizaki Y., Hiraoka N., Obayashi A. Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase. Nucleic Acids Res. 1987 Mar 25;15(6):2653–2664. doi: 10.1093/nar/15.6.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McBride K. E., Summerfelt K. R. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol. 1990 Feb;14(2):269–276. doi: 10.1007/BF00018567. [DOI] [PubMed] [Google Scholar]
  15. McGraw N. J., Bailey J. N., Cleaves G. R., Dembinski D. R., Gocke C. R., Joliffe L. K., MacWright R. S., McAllister W. T. Sequence and analysis of the gene for bacteriophage T3 RNA polymerase. Nucleic Acids Res. 1985 Sep 25;13(18):6753–6766. doi: 10.1093/nar/13.18.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Olins P. O., Devine C. S., Rangwala S. H., Kavka K. S. The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Gene. 1988 Dec 15;73(1):227–235. doi: 10.1016/0378-1119(88)90329-0. [DOI] [PubMed] [Google Scholar]
  17. Palmer J. D. Contrasting modes and tempos of genome evolution in land plant organelles. Trends Genet. 1990 Apr;6(4):115–120. doi: 10.1016/0168-9525(90)90125-p. [DOI] [PubMed] [Google Scholar]
  18. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  19. Shinozaki K., Ohme M., Tanaka M., Wakasugi T., Hayashida N., Matsubayashi T., Zaita N., Chunwongse J., Obokata J., Yamaguchi-Shinozaki K. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986 Sep;5(9):2043–2049. doi: 10.1002/j.1460-2075.1986.tb04464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Staub J. M., Maliga P. Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J. 1993 Feb;12(2):601–606. doi: 10.1002/j.1460-2075.1993.tb05692.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stern D. B., Gruissem W. Control of plastid gene expression: 3' inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell. 1987 Dec 24;51(6):1145–1157. doi: 10.1016/0092-8674(87)90600-3. [DOI] [PubMed] [Google Scholar]
  22. Svab Z., Hajdukiewicz P., Maliga P. Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8526–8530. doi: 10.1073/pnas.87.21.8526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ursin V. M., Irvine J. M., Hiatt W. R., Shewmaker C. K. Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco. Plant Cell. 1991 Jun;3(6):583–591. doi: 10.1105/tpc.3.6.583. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES