Plant Molecular Biology

, Volume 33, Issue 6, pp 989–999 | Cite as

Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage

  • Gerard J.A. Rouwendal*
  • Odette Mendes
  • Emil J.H. Wolbert
  • A. Douwe de Boer


The gene encoding green fluorescent protein (GFP) from Aequorea victoria was resynthesized to adapt its codon usage for expression in plants by increasing the frequency of codons with a C or a G in the third position from 32 to 60%. The strategy for constructing the synthetic gfp gene was based on the overlap extension PCR method using 12 long oligonucleotides as the starting material and as primers. The new gene contains 101 silent nucleotide changes compared to its wild-type counterpart used in this study. Several transgenic tobacco lines containing the wild-type gfp gene contained minute amounts of a smaller protein cross-reacting with GFP antiserum, whereas only one protein of the expected size was found in transgenics with the synthetic gfp gene. The smaller protein was probably encoded by a truncated gfp mRNA created by splicing of a 84 bp cryptic intron as detected by a reverse transcription-PCR technique. A comparison of GFP production in transgenics with the wild-type and the synthetic gfp gene under the control of the enhanced CaMV 35S promoter showed that the large-scale alterations in the gfp gene increased the frequency of high expressors in the transgenic population but hardly changed the maximum GFP concentrations.The latter phenomenon may be attributed to a reduced regeneration capacity of transformed cells with higher GFP concentrations.

transgenic GFP synthetic gene codon usage cryptic intron 


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  1. 1.
    Adang MJ, Brody MS, Cardineau G, Eagan N, Roush RT, Shewmaker CK, Jones A, Oakes JV, McBride KE: The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. PlantMol Biol 21: 1131–1145 (1993).Google Scholar
  2. 2.
    Amann E, Ochs B, Abel K-J: Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69: 301–315 (1988).Google Scholar
  3. 3.
    Baserga SJ, Benz EJ Jr: ß-Globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability. Proc Natl Acad Sci USA 89: 2935–2939 (1992).Google Scholar
  4. 4.
    Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD: Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature 321: 446–449 (1986).Google Scholar
  5. 5.
    Baulcombe DC, Chapman S, Santa Cruz S: Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7: 1045–1053 (1995).Google Scholar
  6. 6.
    Belgrader P, Cheng J, Maquat LE: Evidence to implicate translation by ribosomes in the mechanism by nonsense codons reduce the nuclear level of human triosephoshate isomerase mRNA. Proc Natl Acad Sci USA 90: 482–486 (1993).Google Scholar
  7. 7.
    Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).Google Scholar
  8. 8.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 263: 802–805 (1994).Google Scholar
  9. 9.
    Chambers SP, Prior SE, Bartsow DA, Minton NP: The pMTL nic— cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68: 139–149 (1988).Google Scholar
  10. 10.
    Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J: Engineered GFP as a vital reporter in plants. Curr Biol 6: 325–330 (1996)Google Scholar
  11. 11.
    Cormack BP, Valdivia RH, Falkow S: FACS-optimizedmutants of the green fluorescent protein (GFP). Gene 173: 33–38 (1996)Google Scholar
  12. 12.
    Crameri A, Whitehorn EA, Tate E, Stemmer WPC: Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol 14: 315–319 (1996).Google Scholar
  13. 13.
    Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY: Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20: 448–455 (1995).Google Scholar
  14. 14.
    Del Tito BJ Jr, Ward JM, Hodgson J, Gershater CJL, Edwards H, Wysocki LA, Watson FA, Sathe G, Kane JF: Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli. J Bact 177: 7086–7091 (1995).Google Scholar
  15. 15.
    De Vries SC, Springer J, Wessels JGH: Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings. Planta 156: 129–135 (1982).Google Scholar
  16. 16.
    Düring K, Porsch P, Fladung M, Lörz H: Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant J 3: 587–598 (1993).Google Scholar
  17. 17.
    Gallie DR: Posttranscriptional regulation of gene expression in plants. In: Briggs WR, Jones RL, Walbot V (eds) Annual Review of Plant Physiology and Plant Molecular Biology 44, pp. 77–105. Annual Reviews, Palo Alto (1993).Google Scholar
  18. 18.
    Goodall GJ, Filipowicz W: The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58: 473–483 (1989).Google Scholar
  19. 19.
    Goodall GJ, Filipowicz W: The minimum functional length of pre-mRNA introns in monocots and dicots. Plant Mol Biol 14: 727–733 (1990).Google Scholar
  20. 20.
    Hanley BA, Schuler MA: Plant intron sequences: evidence for distinct groups of introns. Nucl Acids Res 16: 7159–7176 (1988).Google Scholar
  21. 21.
    Haseloff J, Amos B: GFP in plants. Trends Genet 11: 328–329 (1995).Google Scholar
  22. 22.
    Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51–59 (1989).Google Scholar
  23. 23.
    Hoekema A, Kastelein RB, Vassar M, De Boer HA: Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol 7: 2914–2924 (1987).Google Scholar
  24. 24.
    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
  25. 25.
    Hu W, Cheng C-L: Expression of Aequorea green fluorescent protein in plant cells. FEBS Lett 369: 331–334 (1995).Google Scholar
  26. 26.
    Jobling SA, Gehrke L: Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625 (1987).Google Scholar
  27. 27.
    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).Google Scholar
  28. 28.
    Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).Google Scholar
  29. 29.
    Jofuku KD, Schipper RD, Goldberg RB: A frameshift mutation prevents Kunitz trypsin inhibitor mRNA accumulation in soybean embryos. Plant Cell 1: 427–435 (1989).Google Scholar
  30. 30.
    Kay R, Chan A, Daly M, McPherson J: Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302.Google Scholar
  31. 31.
    Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM: Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3: 1187–1193 (1991).Google Scholar
  32. 32.
    Kost B, Schnorf M, Potrykus I, Neuhaus G: Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay. Plant J 8: 155–166 (1995).Google Scholar
  33. 33.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).Google Scholar
  34. 34.
    Mertz M, Rashtchian A: Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNAprobes. Anal Biochem 221: 160–165 (1994).Google Scholar
  35. 35.
    Millar AJ, Short SR, Chua N-H, Kay SA: A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell 4: 1075–1087 (1992).Google Scholar
  36. 36.
    Murray EE, Lotzer J, Eberle M: Codon usage in plant genes. Nucl Acids Res 17: 477–498 (1989).Google Scholar
  37. 37.
    Nagel R, Elliott A, Masel A, Birch RG, Manners JM: Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol Lett 67: 325–328 (1990).Google Scholar
  38. 38.
    Niedz RP, Sussman MR, Satterlee JS: Green fluorescent protein: an in vivo reporter of plant gene expression. Plant Cell Rep 14: 403–406 (1995).Google Scholar
  39. 39.
    Oakes JV, Shewmaker CK, Stalker DM: Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants. Bio/technology 9: 982–986 (1991).Google Scholar
  40. 40.
    Ow DW, Wood KV, DeLuca M, De Wet JR, Helinski DR, Howell S: Transient and stable expression of the firefly luciferase gene in plant cells. Science 234: 856–859 (1986).Google Scholar
  41. 41.
    Perlak FJ, Fuchs RL, Dean DA, McPherson SA, Fischhoff DA: Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88: 3324–3328 (1991).Google Scholar
  42. 42.
    Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ: Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111: 229–233 (1992).Google Scholar
  43. 43.
    Rosenberg AH, Goldman E, Dunn JJ, Studier FW, Zubay G: Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bact 175: 716–722 (1993).Google Scholar
  44. 44.
    Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW: Green-fluorescent protein as a new vital marker in plant cells. Plant J 8: 777–784 (1995).Google Scholar
  45. 45.
    Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77: 5201–5205 (1980).Google Scholar
  46. 46.
    van Aarssen R, Soetaert P, Stam M, Dockx J, Gosselé V, Seurinck J, Reynaerts A, Cornelissen M: cryIA(b) transcript formation in tobacco is inefficient. Plant Mol Biol 28: 513–524 (1995).Google Scholar
  47. 47.
    Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M: Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250 (1990).Google Scholar
  48. 48.
    Vancanneyt G, Rosahl S, Willmitzer L: Translatability of a plant-mRNA influences its accumulation in transgenic plants. Nucl Acids Res 18: 2917–2921 (1990).Google Scholar
  49. 49.
    Waigmann E, Barta A: Processing of chimeric introns in dicot plants: evidence for a close cooperation between 5′ and 3′ splice sites. Nucl Acids Res 20: 75–81 (1992).Google Scholar
  50. 50.
    Wang S, Hazelrigg T: Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Science 369: 400–403 (1994).Google Scholar
  51. 51.
    Yanisch-Perron C, Vieira J, Messing J: Improved M13 cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33: 103–119 (1985).Google Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Gerard J.A. Rouwendal*
    • 1
  • Odette Mendes
    • 1
  • Emil J.H. Wolbert
    • 1
  • A. Douwe de Boer
    • 1
  1. 1.Department of Plant Molecular Regulation and QualityAgrotechnological Research Institute (ATO-DLO)WageningenNetherlands

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