Skip to main content
SpringerLink
Log in

Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene from Bacillus thuringiensis with the Neurotoxin Gene hwtx-I

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC50 (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Aronson A (2002) Sporulation and delta-endotoxin synthesis by Bacillus thuringiensis. Cell Mol Life Sci 59:417–425

    Article  PubMed  CAS  Google Scholar 

  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Short protocols in molecular biology, 3rd edn. John Wiley & Sons, New York

    Google Scholar 

  3. Bosch D, Schipper B, Kleij D, Maagd RA, Stiekema WJ (1994) Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Biotechnology 12:915–918

    Article  PubMed  CAS  Google Scholar 

  4. Chang L, Grant R, Aronson A (2001) Regulation of the packaging of Bacillus thuringiensis δ-endotoxins into inclusions. Appl Environ Microbiol 67:5032–5036

    Article  PubMed  CAS  Google Scholar 

  5. Chen XJ, Lee MK, Dean DH (1993) Site-directed mutations in a highly conserved region of Bacillus thuringiensis δ-endotoxin affect inhibition of short circuit current across Bombyx mori midgets. Proc Natl Acad Sci USA 90:9041–9045

    Article  PubMed  CAS  Google Scholar 

  6. Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813

    CAS  Google Scholar 

  7. Ding XZ, Liu QL, Mo XT, Gao BD, Xia LQ (2003) Characterization of insecticidal crystal proteins genes from Bacillus thuringiensis 4.0718 strain. Acta Microbiol Sinica 43:413–417

    CAS  Google Scholar 

  8. Ding XZ, Luo ZH, Xia LQ, Gao BD, Sun YJ, Zhang YM (2008) Improving the insecticidal activity by expression of a recombinant cry1Ac gene with chitinase-encoding gene in acrystalliferous Bacillus thuringiensis. Curr Microbiol 56:442–446

    Article  PubMed  CAS  Google Scholar 

  9. Guo SD, Cui HZ, Xia LQ, Wu DL, Ni WZ, Zhang ZL, Zhang BL, Xu YJ (1999) Study on double-effect resistant insects transgenic cotton. Sci Agric Sinica 32:1–7

    Google Scholar 

  10. Guo SD, Cui HZ, Xia LQ et al (1999) Development of bivalent insect-resistant transgenic cotton plants. Sci Agric Sinica 32(3):1–7

    Google Scholar 

  11. Li M, Li LY, Wu X, Liang SP (2000) Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena). Toxicon 38:153–162

    Article  PubMed  Google Scholar 

  12. Liang SP, Zhang DY, Pan X, Chen Q, Zhou PA (1993) Properties and amino acid sequence of tuwentoxin-1, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia huwena. Toxicon 31:969–978

    Article  PubMed  CAS  Google Scholar 

  13. Liu YB, Tabashnik BE, Denneby TJ et al (1999) Development time and resistance to Bacillus thuringiensis crops. Nature 400:501–502

    Article  Google Scholar 

  14. MacIntosh SC, Stone TB, Sims SR, Hunst PL, Greenplate JT, Marrone PG, Perlak FJ, Fischhoff DA, Fuchs RL (1990) Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects. J Invertebr Pathol 56:258–266

    Article  PubMed  CAS  Google Scholar 

  15. Morán R, García R, López A, Zaldúa Z, Mena J, García M, Armas R, Somonte D, Rodríguez J, Gómez M, Pimentel E (1998) Transgenic sweet potato plants carrying the delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis. Plant Sci 139:175–184

    Article  Google Scholar 

  16. Neirlich DP, Murakawa GJ (1996) The decay of bacterial messenger RNA. Prog Nucleic Acid Res Mol Biol 52:153–216

    Article  Google Scholar 

  17. Qu Y, Liang S, Ding J, Ma L, Zhang R, Gu X (1995) Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spider Selenocosmia huwena: 1. Sequence-specific 1H-NMR assignments. J Protein Chem 14:549–557

    Article  PubMed  CAS  Google Scholar 

  18. Palidam M (1992) The insecticidal crystal protein Cry1A(c) from Bacillus thuringiensis in highly toxic for Heliothis armigera. J Invertebr Pathol 59:109–111

    Article  Google Scholar 

  19. Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938

    PubMed  CAS  Google Scholar 

  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  21. Wong HC, Schnepf HE, Whiteley HR (1983) Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem 258:1960–1967

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the National Natural Science Foundation of China (No. 30670052 and 30870064), the National 863 Project of China (Nos. 2006AA02Z187 and 2006AA10A212), the Research Fund for the Doctoral Program of Higher Education (No. 20060542006), and the Provincial Natural Science Foundation of Hunan (No. 06JJ2009).

Author information

Authors and Affiliations

  1. College of Life Sciences of Hunan Normal University, Key Laboratory of Microbial Molecular Biology, Changsha, 410081, Hunan Province, China

    LiQiu Xia, XiaoShan Long, XueZhi Ding & YouMing Zhang

Authors
  1. LiQiu Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. XiaoShan Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. XueZhi Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. YouMing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to LiQiu Xia.

Additional information

LiQiu Xia and XiaoShan Long contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xia, L., Long, X., Ding, X. et al. Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene from Bacillus thuringiensis with the Neurotoxin Gene hwtx-I . Curr Microbiol 58, 52–57 (2009). https://doi.org/10.1007/s00284-008-9265-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-008-9265-y

Keywords

Search

Navigation