Plant Molecular Biology

, Volume 16, Issue 1, pp 1–10

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

  • Yuji Fukuda
  • Masaru Ohme
  • Hideaki Shinshi
Article

Abstract

We have isolated and characterized the genomic clone λCHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on λCHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5′ upstream region fused to the coding sequence of β-glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5′ deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.

Key words

chimeric gene chitinase gene gene family promoter region transient expression 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    BentonWD, DavisRW: Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182 (1977).Google Scholar
  2. 2.
    BollerT: Induction of hydrolases as a defense reaction against pathogens. In: KeyJL, KosugeT (eds) Cellular and Molecular Biology of Plant Stress, pp. 247–262. Alan R. Liss, New York (1985).Google Scholar
  3. 3.
    BradfordMM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).Google Scholar
  4. 4.
    BreathnachR, ChambonP: Organization and expression of eucaryotic split genes coding for proteins. Ann Rev Biochem 50: 349–383 (1981).Google Scholar
  5. 5.
    BroglieKE, GaynorJJ, BroglieRM: Ethylene-regulated gene expression: Molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris. Proc Natl Acad Sci (USA) 83: 6820–6824 (1986).Google Scholar
  6. 6.
    BroglieKE, BiddleP, CressmanR, BroglieRM: Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco. Plant Cell 1: 599–607 (1989).Google Scholar
  7. 7.
    ChurchGM, GilbertW: Genomic sequencing. Proc Natl Acad Sci (USA) 81: 1991–1995 (1984).Google Scholar
  8. 8.
    CoureyAJ, TjianR: Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55: 887–898 (1988).Google Scholar
  9. 9.
    Durand-TardifM: Etude de l'induction, par l'ethephon, de l'expression du gène codant pour la chitinase chez la tomate et analyse de la structure de ce gène. Doctoral Dissertation, Université de Paris Sud, Paris (1986).Google Scholar
  10. 10.
    FeinbergAP, VogelsteinB: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).Google Scholar
  11. 11.
    FelixG, MeinsFJr: Ethylene regulation of β-1,3-glucanase in tobacco. Planta 172: 386–392 (1987).Google Scholar
  12. 12.
    FrischaufA-M, LehrachH, PoustkaA, MurrayN: Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170: 827–842 (1983).Google Scholar
  13. 13.
    HedrickSA, BellJN, BollerT, LambCJ: Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection. Plant Physiol 86: 182–186 (1988).Google Scholar
  14. 14.
    Hooft van HuijsduijnenRAM, KauffmannS, BrederodeFTh, CornelissenBJC, LegrandM, FritigB, BolJF: Homology between chitinases that are induced by TMV infection of tobacco. Plant Mol Biol 9: 411–420 (1987).Google Scholar
  15. 15.
    JametE, DurrA, FleckJ: Absence of some truncated genes in the amphidiploid Nicotiana tabacum. Gene 59: 213–221 (1987).Google Scholar
  16. 16.
    JeffersonRA, BurgessSM, HirshD: β-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci (USA) 83: 8447–8451 (1986).Google Scholar
  17. 17.
    JeffersonRA, KavanaghTA, BevanMW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).Google Scholar
  18. 18.
    JofukuKD, GoldbergRB: Analysis of plant gene structure. In: ShawCH (ed) Plant molecular biology: a practical approach, pp. 37–66. IRL Press, Washington DC (1988).Google Scholar
  19. 19.
    LegrandM, KauffmannS, GeoffroyP, FritigB: Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases. Proc Natl Acad Sci (USA) 84: 6750–6754 (1987).Google Scholar
  20. 20.
    LogemannJ, SchellJ, WillmitzerL: Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20 (1987).Google Scholar
  21. 21.
    ManiatisT, FritschEF, SambrookJ: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  22. 22.
    MauchF, HadwigerLA, BollerT: Ethylene: Symptom, not signal for the induction of chitinase and β-1,3-glucanase in pea pods by pathogens and elicitors. Plant Physiol 76: 607–611 (1984).Google Scholar
  23. 23.
    MurashigeT, SkoogF: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).Google Scholar
  24. 24.
    NagataT, OkadaK, TakebeI, MatsuiC: Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol Gen Genet 184: 161–165 (1981).Google Scholar
  25. 25.
    ReedKC, MannDA: Rapid transfer of DNA from agarose gels to nylon membranes. Nucl Acids Res 13: 7207–7271 (1985).Google Scholar
  26. 26.
    RobyD, ToppanA, Esquerré-TugayéM: Cell surfaces in plant-microorganism interactions. Plant Physiol 81: 228–233 (1986).Google Scholar
  27. 27.
    SangerF, NicklenS, CoulsonAR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci (USA) 74: 5463–5467 (1977).Google Scholar
  28. 28.
    ShinshiH, MohnenD, MeinsFJr: Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci (USA) 84: 89–93 (1987).Google Scholar
  29. 29.
    Shinshi H, Neuhaus J-M, Ryals J, Meins F Jr: Structure of a tobacco endochitinase gene: Evidence that different chitinase genes can arise by transposition of sequences encoding a cystein-rich domain. Plant Mol Biol 14: 357–368.Google Scholar
  30. 30.
    TongCB, LabavitchJM, YangSF: The induction of ethylene production from pear cell culture by cell wall fragments. Plant Physiol 81: 929–930 (1986).Google Scholar
  31. 31.
    Vögeli-LangeR, Hansen-GehriA, BollerT, MeinsFJr: Induction of the defense-related glucanohydrolases β-1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves. Plant Sci 54: 171–176 (1988).Google Scholar
  32. 32.
    WatanabeY, MeshiT, OkadaY: Infection of tobacco protoplasts with in vitro transcribed tobacco mosaic virus RNA using an improved electroporation method. FEBS Lett 219: 65–69 (1987).Google Scholar
  33. 33.
    Yanisch-PerronC, VieiraJ, MessingJ: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119 (1985).Google Scholar

Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • Yuji Fukuda
    • 1
  • Masaru Ohme
    • 1
  • Hideaki Shinshi
    • 1
  1. 1.Fermentation Research InstituteAgency of Industrial Science and TechnologyTsukuba, IbarakiJapan

Personalised recommendations