Skip to main content

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice, Confers Resistance to the Rice Weevil Sitophilus Oryzae

Abstract

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R3 seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.

This is a preview of subscription content, access via your institution.

References

  • Alfonso-Rubi J, Carbonero P and Diaz I (1999) Parameters influencing the regeneration capacity of calluses derived from mature indica and japonica seeds after microprojectile bombardment. Euphytica 107: 115–122.

    Google Scholar 

  • Alfonso-Rubi J, Ortego F, Sanchez-Monge R, Garcia-Casado G, Pujol M, Castañera P et al. (1997) Wheat and barley inhibitors active towards α-amylase and trypsin-like activities from Spodoptera frugiperda. J Chem Ecol 23: 1729–1741.

    Google Scholar 

  • Altpeter F, Díaz I, McAuslane H, Gaddour K, Carbonero P and Vasil IK (1999) Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor Cme. Mol Breed 5: 53–63.

    Google Scholar 

  • Baker JE (1982) Digestive proteinases of Sitophilus weevil (Coleoptera: Curculionidae) and their response to inhibitors from wheat and corn flour. Can J Zool 60: 3206–3214.

    Google Scholar 

  • Balachowsky AS (1963) Entomologie Appliquée a L'Agriculture. Tome I, Coléoptères, Masson et Cie Eds, Paris.

  • Bonadé-Bottino M, Lerin J, Zaccomer B and Jouanin L (1999) Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystain I. Insect Biochem Mol Biol 29: 131–138.

    Google Scholar 

  • Carbonero P, Diaz I, Vicente-Carbajosa J, Alfonso-Rubi J, Gaddour K and Lara P (1999) Cereal α-amylase/trypsin inhibitors and transgenic insect resistance. In: Scarascia-Mugnozza GI, Porceddu E and Pagnotta MA (eds), Genetics and Breeding for Crop Quality and Resistance. (pp. 147–158) Kluwer Academic Publishers.

    Google Scholar 

  • Chen L, Zhang S, Beachy RN and Fauquet CM (1998) A protocol for consistent, large scale production of fertile transgenic rice plants. Plant Cell Rep 18: 25–31.

    Google Scholar 

  • Duan X, Li X, Xue Q, Abo-El-Saad M, Xu D and Wu R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotech 14: 494–498.

    Google Scholar 

  • Elden TC (1995) Selected proteinase inhibitor effects on alfalfa weevil (Coleoptera: Curculionidae) growth and development. J Econ Entomol 88: 1586–1590.

    Google Scholar 

  • Giri CC and Laxmi GV (2000) Production of transgenic rice with agronomically useful genes: an assessment. Biotechnol Adv 18: 653–683.

    Google Scholar 

  • Hilder VA and Boulter D (1999) Genetic engineering of crop plants for insect resistance-a critical review. Crop Prot 18: 177–191.

    Google Scholar 

  • Irie K, Hosoyama H, Takeuchi T, Iwabuchi K, Watanabe H, Abe M et al. (1996) Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. Plant Mol Biol 30: 149–157.

    Google Scholar 

  • IRRI (2000) World Rice Statistics. International Rice Research Institute.

  • Jouanin L, Bonade-Bottino M, Girard C, Morrot G and Giband M (1998) Transgenic plants for insect resistance. Plant Sci 131: 1–11.

    Google Scholar 

  • Krattiger AF (1997) A case study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs 2: 42, ISAAA, Ithaca, NY

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277: 680–685.

    Google Scholar 

  • Lara P, Ortego F, González-Hidalgo E, Castañera P, Carbonero P and Diaz I (2000) Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco. Transgenic Res 9: 169–178.

    Google Scholar 

  • Lee AI, Lee S-H, Koo JC, Chum HJ, Lim CO, Mun JH et al. (1999) Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stal) in transgenic rice. Mol Breed 5: 1–9.

    Google Scholar 

  • Lee T-M and Lin Y-H (1995) Trypsin inhibitor and trypsin-like activity in air-or submergence-grown rice (Oryza sativa L.) coleoptiles. Plant Sci 106: 43–54.

    Google Scholar 

  • Liang C, Brookhart G, Feng GH, Reeck GR and Kramer KJ (1991) Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed. FEBS Lett 278: 139–142.

    Google Scholar 

  • Maagd RA, Bosch D and Stikema W (1999) Bacillus thuringiensis toxin-mediated insect resistance in plants. Trends Plant Sci 4: 9–13.

    Google Scholar 

  • Maagd RA, Bravo A and Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17: 193–199.

    Google Scholar 

  • Maqbool SB, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA and Christou P (2001) Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol Breed 7: 85–93.

    Google Scholar 

  • Michaud D, Faye L and Yelle S (1993) Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis 14: 94–98.

    Google Scholar 

  • Mochizuki A, Nishizawa Y, Onodera H, Tabei Y et al. (1999) Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis. Entomol Exp Appl 93: 173–178.

    Google Scholar 

  • Ortego F, Farinós GP, Ruíz M, Marco V and Castañera P (1998) Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival. Entomol Exp Appl 88: 265–274.

    Google Scholar 

  • Pittendrigh BR, Huesing JE, Shade RE and Murdock LL (1997) Effects of lectins, CRY1A/CRY1B Bt d-endotoxin, PAPA, protease and α-amylase inhibitors, on the development of the rice weevil, Sitophilus oryzae, using an artificial seed bioassay. Entomol Exp Appl 82: 201–211.

    Google Scholar 

  • Rakwal R, Agrawal GK and Jwa N-S (2001) Characterization of a rice (Oryza sativa L.) Bowman-Birk proteinase inhibitor: tightly light regulated induction in response to cut, jasmonic acid, ethylene and protein phosphatase 2A inhibitors. Gene 262: 189–198.

    Google Scholar 

  • Rodriguez-Palenzuela P, Royo J, Gómez L, Sánchez-Monge R, Salcedo G, Molina-Cano JL et al. (1989) The gene for trypsin inhibitor CMe is regulated in trans by the lys3a locus in the endosperm of barley (Hordeum vulgare L.). Mol Gen Genet 219: 474–479.

    Google Scholar 

  • Royo J, Diaz I, Rodriguez-Palenzuela P and Carbonero P (1996) Isolation and promoter characterization of barley gene Itr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutant Lys3a endosperm. Plant Mol Biol 31: 1051–1059.

    Google Scholar 

  • Sambrook J, Fritsch EF and Maniatis T (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  • Schuler TH, Poppy GM, Kerry BR and Denholm I (1998) Insectresistant transgenic plants. Trends Biochem Sci 16: 168–175.

    Google Scholar 

  • Taylor B and Powell A (1988) Isolation of plant DNA and RNA. Focus 4: 4–6.

    Google Scholar 

  • Tyagi AK and Mohanty A (2000) Rice transformation for crop improvement and functional genomics. Plant Sci 158: 1–18.

    Google Scholar 

  • Wüun J, Klöti A, Burkhardt PK, Ghosh GC, Launis K, Iglesias VA et al. (1996) Transgenic indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control. Biotechnol 14: 171–176.

    Google Scholar 

  • Xu D, Xue Q, McElroy D, Mawai Y, Líder V and Wu R (1996) Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major insect pests. Mol Breed 2: 167–173.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Isabel Díaz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Alfonso-Rubí, J., Ortego, F., Castañera, P. et al. Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice, Confers Resistance to the Rice Weevil Sitophilus Oryzae . Transgenic Res 12, 23–31 (2003). https://doi.org/10.1023/A:1022176207180

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1022176207180

  • barley trypsin inhibitor
  • Sitophilus oryzae
  • storage pest resistance
  • transgenic rice