Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1987 Dec;85(4):1103–1109. doi: 10.1104/pp.85.4.1103

Bacillus thuringiensis §-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects

Kenneth A Barton 1,2, H R Whiteley 1,2, Ning-Sun Yang 1,2
PMCID: PMC1054402  PMID: 16665812

Abstract

The crystal proteins, or §-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.

Full text

PDF

Images in this article

1105Fig. 2
on p.1105
1106Fig. 3
on p.1106
1107Fig. 4
on p.1107
1107Fig. 5
on p.1107
1108Fig. 6
on p.1108

Selected References

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

  1. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  2. Aronson A. I., Beckman W., Dunn P. Bacillus thuringiensis and related insect pathogens. Microbiol Rev. 1986 Mar;50(1):1–24. doi: 10.1128/mr.50.1.1-24.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barton K. A., Binns A. N., Matzke A. J., Chilton M. D. Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell. 1983 Apr;32(4):1033–1043. doi: 10.1016/0092-8674(83)90288-x. [DOI] [PubMed] [Google Scholar]
  4. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H. M. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1982;1(6):561–573. [PubMed] [Google Scholar]
  8. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987 Apr 24;15(8):3257–3273. doi: 10.1093/nar/15.8.3257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gardner R. C., Howarth A. J., Hahn P., Brown-Luedi M., Shepherd R. J., Messing J. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucleic Acids Res. 1981 Jun 25;9(12):2871–2888. doi: 10.1093/nar/9.12.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gehrke L., Auron P. E., Quigley G. J., Rich A., Sonenberg N. 5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation. Biochemistry. 1983 Oct 25;22(22):5157–5164. doi: 10.1021/bi00291a015. [DOI] [PubMed] [Google Scholar]
  11. Hood E. E., Helmer G. L., Fraley R. T., Chilton M. D. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol. 1986 Dec;168(3):1291–1301. doi: 10.1128/jb.168.3.1291-1301.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. John M. E., John M. C., Ashley P., MacDonald R. J., Simpson E. R., Waterman M. R. Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5628–5632. doi: 10.1073/pnas.81.18.5628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ow D. W., Jacobs J. D., Howell S. H. Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4870–4874. doi: 10.1073/pnas.84.14.4870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peralta E. G., Hellmiss R., Ream W. Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J. 1986 Jun;5(6):1137–1142. doi: 10.1002/j.1460-2075.1986.tb04338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  17. Schnepf H. E., Whiteley H. R. Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene. J Biol Chem. 1985 May 25;260(10):6273–6280. [PubMed] [Google Scholar]
  18. Schnepf H. E., Wong H. C., Whiteley H. R. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem. 1985 May 25;260(10):6264–6272. [PubMed] [Google Scholar]
  19. Whiteley H. R., Schnepf H. E. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annu Rev Microbiol. 1986;40:549–576. doi: 10.1146/annurev.mi.40.100186.003001. [DOI] [PubMed] [Google Scholar]
  20. Yadav N. S., Vanderleyden J., Bennett D. R., Barnes W. M., Chilton M. D. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6322–6326. doi: 10.1073/pnas.79.20.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES