World Journal of Microbiology and Biotechnology

, Volume 8, Issue 6, pp 638–644 | Cite as

Enhanced stable expression of aVibrio luciferase under the control of the Ω-translational enhancer in transgenic plants

  • K. Okumura
  • L. Chlumsky
  • T. O. Baldwin
  • C. I. Kado
Research Papers


A fusion gene usingluxA andluxB genes ofVibrio species has been designed to express light autonomously in plants.LuxA:luxB was introduced into plants by a high-efficiency transformation system consisting of a high-copy virulence helper plasmid pUCD2614 and T-vector pUCD2715 containingluxA:luxB. The expression ofluxA:luxB fusion gene was optimized by adjusting the spacing between the genes and by placing the translational efficiency of its mRNA under the control of the Ω-3 translational enhancer. The resulting transgenic plants synthesized luciferase at levels greater than 1% of the total leaf protein. These plants produced light autonomously and light intensity was enhanced by the addition of aldehyde. That theluxA:luxB fusion has been optimized enables its use as a reporter for gene activity in plants during development and under various stress-inducing conditions. These results show that a specific protein from an introduced foreign gene can be produced with high efficiency in cultivated plants and such a system is therefore amenable for production of desired proteins through conventional farming methods.

Key words

Agrobacterium bioluminescence high-copyvir vector high-efficiency protein production high-efficiency transformation of plants T-vectors Vibrio fischeri Vibrio harveyi 


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  1. Baldwin, T.O., Devine, J.H., Heckel, R.C., Lin, J.-W., Shadel, G.S. 1989 The complete nucleotide sequence of thelux regulon ofVibrio fischeri and theluxABN region ofPhotobacterium leiognathi and the mechanism of control of bioluminescence.Journal of Bioluminescence and Chemiluminescence 4, 326–341.PubMedGoogle Scholar
  2. Baldwin, T.O. & Zeigler, M.M. 1991 The biochemistry and molecular biology of bacterial bioluminescence. InChemistry and Biochemistry of Flavoenzymes, Vol. 3, ed Muller, F. Boca Raton, FL: CRC Press.Google Scholar
  3. Belas, R., Mileham, A., Cohn, D., Hilmen, M., Simon, M. & Silverman, M. 1982 Bacterial bioluminescence: isolation and expression of the luciferase genes fromVibrio harveyi.Science 218, 791–793.PubMedGoogle Scholar
  4. Benfey, P.N. & Chua, N.H. 1990. The cauliflower mosaic virus 35S promoter: combinational regulation of transcription in plants.Science 250, 959–966.Google Scholar
  5. Bradford, M.M. 1976 A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry 72, 248–254.PubMedGoogle Scholar
  6. Brasier, A.R., Tate, J.E. & Habener, J.F. 1989 Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines.Biotechniques 7, 1116–1122.PubMedGoogle Scholar
  7. Cooley, M.B., D'Souza, M.R. & Kado, C.I 1991 ThevirC andvirD operons of theAgrobacterium Ti plasmid are regulated by theros chromosomal gene: analysis of the clonedros gene.Journal of Bacteriology 173, 2608–2616.PubMedGoogle Scholar
  8. Dale, E.C. & Ow, D.W. 1991 Gene transfer with subsequent removal of the selection gene from the host genome.Proceedings of the National Academy of Sciences of the United States of America 88, 10558–10562.PubMedGoogle Scholar
  9. DeWeger, L.A., Dunbar, P., Mahafee, W.F., Lugtenberg, B.J.J. & Sayler, G.S. 1991 Use of bioluminescence markers to detectPseudomonas spp. in the rhizosphere.Applied and Environmental Microbiology 57, 3641–3644.Google Scholar
  10. De Wet, J., Wood, K.V., DeLuca, M., Helinski, D.R. & Subramani, S. 1987 Firefly luciferase gene: structure and expression in mammalian cells.Molecular and Cellular Biology 7, 725–737.PubMedGoogle Scholar
  11. Engebrecht, J., Nealson, K. & Silverman, M. 1983 Bacterial bioluminescence: isolation and genetic analysis of functions fromVibrio fischeri.Cell 32, 773–781.PubMedGoogle Scholar
  12. Engebrecht, J., Simon, M. & Silverman, M. 1985 Measuring gene expression with light.Science 227, 1345–1347.PubMedGoogle Scholar
  13. Gallie, D.R. & Kado, C.I. 1989 A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence.Proceedings of the National Academy of Science of the United States of America 86, 129–132.Google Scholar
  14. Gallie, D.R., Kado, C.I., Hershey, J.W.B., Wilson, M.A. & Walbot, V. 1989 Eukaryotic viral 5′-leader sequences act as translational enhancers in eukaryotes and prokaryotes. InMolecular Biology of RNA, ed Cech, T.R. pp. 237–256. New York: Alan R. Liss.Google Scholar
  15. Hirooka, T., Rogowsky, P.M. & Kado, C.I. 1987 Characterization of thevirE locus ofAgrobacterium tumefaciens plasmid pTiC58.Journal of Bacteriology 169, 1529–1536.PubMedGoogle Scholar
  16. Hirooka, T., Shaw, J.J. & Kado, C.I. 1988 Use of bioluminescence in the field of plant pathology.Syokubutsu-Boeki Journal 10, 1–6.Google Scholar
  17. Horsch, R.B. & Klee, H.J. 1986 Rapid assay of foreign gene expression in leaf discs transformed byAgrobacterium tumefaciens: role of T-DNA borders in the transfer process.Proceedings of the National Academy of Sciences of the United States of America 83, 4428–4432.Google Scholar
  18. Kado, C.I. 1992 Lux and other reporter genes. InMicrobial Ecology. Principles, Methods, and Applications, eds Levin, M.A., Seidler, R.J. & Rogul, M. pp. 371–392. New York: McGraw-Hill.Google Scholar
  19. Kado, C.I. & Liu, S.-T. 1981 Rapid procedure for detection and isolation of large and small plasmids.Journal of Bacteriology 145, 1365–1373.PubMedGoogle Scholar
  20. Kado, C.I. 1990 A glowing christmas tree. InSpirit of Enterprise: The 1990 Rolex Awards, ed Reed, D.W. pp. 199–201. Bern, Buri Druck.Google Scholar
  21. Kamoun, S. & Kado, C.I. 1990a A plant-inducible gene ofXanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts.Journal of Bacteriology 172, 5165–5172.PubMedGoogle Scholar
  22. Kamoun, S. & Kado, C.I. 1990b Phenotypic switching affecting chemotaxis xanthan production, and virulence inXanthomonas campestris.Applied and Environmental Microbiology 56, 3855–3860.Google Scholar
  23. Kamoun, S., Kamdar, H.V., Tola, E. & Kado, C.I. 1992 Incompatible interactions between crucifers andXanthomonas campestris involve a vascular hypersensitive response: role of thehrpX locus.Molecular Plant-Microbe Interactions 5, 22–33.Google Scholar
  24. King, J.M.H., DiGrazia, P.M., Appleate, B., Burlage, R., Sanseverino, J., Dunbar, P., Laimer, F. & Sayler, S.G. 1990 Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation.Science 249, 778–781.Google Scholar
  25. Kirchner, G., Roberts, J.L., Gustafon, G.D., & Ingolia, T.D. 1989 Active bacterial luciferase from a fused gene: expression of aVibrio harveyi luxAB translational fusion in bacteria, yeast and plant cells.Gene 81, 349–354.PubMedGoogle Scholar
  26. Klapwijk, P.M., van Beelen, P. & Schilperoort, R.A. 1979 Isolation of a recombination deficientAgrobacterium tumefaciens mutant.Molecular and General Genetics 173, 171–175.PubMedGoogle Scholar
  27. Koncz, C., Olsson, O., Langridge, W.H.R., Schell, J. & Szalay, A.A. 1987 Expression and assembly of functional bacterial luciferase in plants.Proceedings of the National Academy of Sciences of the United States of America 84, 131–135.Google Scholar
  28. Maniatis, T., Fritsch, E.F. & Sambrook, J. 1982Molecular Cloning. A Laboratory Manual. New York: Cold Spring Harbor Press.Google Scholar
  29. Meighen, E.A. 1991 Molecular biology of bacterial bioluminescence.Microbiological Reviews 55, 123–142.PubMedGoogle Scholar
  30. Mettler, I. 1987 A simple and rapid method for minipreparation of DNA from tissue cultured plant cells.Plant Molecular Biology Reporter 5, 346–349.Google Scholar
  31. Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures.Physiologia Plantarum 15, 473–497.Google Scholar
  32. Nguyen, V.T., Morange, M. & Bensaude, O. 1989 Protein denaturation during heat shock stress.Escherichia coli β-galactosidase andPhotinus pyralis luciferase inactivation in mouse cells.Journal of Biological Chemistry 264, 10487–10492.PubMedGoogle Scholar
  33. Ow, D.W., Wood, K.V., DeLuca, M., DeWet, J., Helinski, D. & Howell, S.H. 1986 Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants.Science 234, 856–859.Google Scholar
  34. Ow, D.W., Jacobs, J.D. & Howell, S.H. 1987 Functional regions of the cauliflower virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity.Proceedings of the National Academy of Sciences of the United States of America 84, 4870–4874.Google Scholar
  35. Palomares, A.J., DeLuca, M. & Helinski, D.R. 1989 Firefly luciferase as a reporter enzyme for measuring gene expression in vegetative and symbioticRhizobium meliloti and other Gram-negative bacteria.Gene 81, 55–64.PubMedGoogle Scholar
  36. Quayle, T.J.A., Hetz, W. & Feix, G. 1991 Characterization of a maize endosperm culture expressing zein genes and its use in transient transformation assays.Plant Cell Reports 9, 544–548.Google Scholar
  37. Radloff, R., Bauer, W. & Vinograd, J. 1967 A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in Hela cells.Proceedings of the National Academy of Sciences of the United States of America 57, 1514–1521.PubMedGoogle Scholar
  38. Riggs, C.D., Hunt, D.C., Lin J. & Chrispeels, M.J. 1989 Utilization of luciferase fusion genes to monitor differential regulation of phytohemagglutinin and phaseolin in promoters in transgenic tobacco.Plant Sciences 63, 47–57.Google Scholar
  39. Rogowsky, P.M., Close, T.J., Chimera, J.A., Shaw, J.J. & Kado, C.I. 1987 Regulation of thevir genes ofAgrobacterium tumefaciens.Journal of Bacteriology 169, 5101–5112.PubMedGoogle Scholar
  40. Rogowsky, P.M., Powell, B.S., Shirasu, K., Lin, T.-S., Morel, P., Zyprian, E., Steck, T.R. & Kado, C.I. 1990 Molecular characterization of thevir regulon ofAgrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 38.63-kbp regulon cloned as a single unit.Plasmid 23, 85–106.PubMedGoogle Scholar
  41. Shaw, J.J., Close, T.J., Engebrecht, J. & Kado, C.I. 1985 Use of bioluminescence to monitorAgrobacterium, Erwinia, Pseudomonas andXanthomonas in plants.Phytopathology 75, 1288.Google Scholar
  42. Shaw, J.J. & Kado, C.I. 1986 Development of aVibrio bioluminescent gene-set to monitor phytopathogenic bacteria during the ongoing disease process in a non-disruptive manner.Bio/Technology 4, 560–564.Google Scholar
  43. Shaw, J.J., Settles, L.G. & Kado, C.I. 1988 TransposonTn4431 mutagenesis ofXanthomonas campestris pv. campestris: characterization of a nonpathogenic mutant and cloning of a locus for pathogenicity.Molecular Plant-Microbe Interactions 1, 39–45.Google Scholar
  44. Shaw, J.J., Dane, F., Geiger, D. & Kloepper, J.W. 1992 Use of bioluminescence for detection of genetically engineered microorganisms released into the environment.Applied and Environmental Microbiology 58, 267–273.PubMedGoogle Scholar
  45. Silverman, M., Martin, M. & Engebrecht, J. 1989 Regulation of luminescence in marine bacteria. InGenetics of Bacterial Diversity, eds Hopwood, D.A. & Chater, K.E. pp. 71–86. London: Academic Press.Google Scholar
  46. Tamiya, E., Sugiyama, T., Masaki, K., Hirose, A., Okoshi, T. & Karube, I. 1990 Spacial imaging of luciferase gene expression in transgenic fish.Nucleic Acids Research 18, 1072.PubMedGoogle Scholar
  47. Thompson, E.M., Nagata, S. & Tsuji, F.I. 1990Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells.Gene 96, 257–262.PubMedGoogle Scholar
  48. Wahl, G.M., Stern, M. & Stark, G.R. 1979 Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.Proceedings of the National Academy of Sciences of the United States of America 76, 3683–3687.PubMedGoogle Scholar
  49. Zyprian, E. & Kado, C.I. 1990Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copyvir helper plasmid.Plant Molecular Biology 15, 245–256.PubMedGoogle Scholar

Copyright information

© Rapid Communications of Oxford Ltd 1992

Authors and Affiliations

  • K. Okumura
    • 1
  • L. Chlumsky
    • 2
  • T. O. Baldwin
    • 2
  • C. I. Kado
    • 1
  1. 1.Department of Plant PathologyUniversity of CaliforniaDavisUSA
  2. 2.Department of Biochemistry and BiophysicsTexas A & M UniversityCollege StationUSA

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