Science in China Series C: Life Sciences

, Volume 48, Issue 4, pp 346–356 | Cite as

Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

  • Guirong Wang
  • Kongming Wu
  • Gemei Liang
  • Yuyuan Guo
Article
  • 78 Downloads

Abstract

Cadherins belong to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is related to insect’s resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmembrane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respectively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recombinant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which located between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence, suggesting that the decrease of expression quantity of BtR-harm is one of the main reasons for H. armigera resistance to Cry1Ac.

Keywords

BtR-harm Bt receptor gene cloning and expression Helicoverpa armigera 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Yu, Z. N., Bacillus thuringiensis (in Chinese), Beijing: Science Press, 1990.Google Scholar
  2. 2.
    Schnepf, E., Crickmore, N., Van Rie J. et al., Bacillus thuringiensis and its pesticidal crystal protein, Microbiol. Mol. Biol. Rev., 1998, 62:755–806.Google Scholar
  3. 3.
    Zhang, B.H., Feng, R., ed., Cotton Insect Resistance and Insect-resistant Cotton (in Chinese), Beijing: China Agricultural Science Press, 2000, 306–326.Google Scholar
  4. 4.
    Jia, S. R., Guo, S. D., An, D. C., ed., Transgenic Cotton (in Chinese), Beijing: Science Press, 2001, 15–57.Google Scholar
  5. 5.
    Huang, D.F., Lin, M., ed., Gene Engineering of Agricultural Microorganism, Beijing: Science Press, 2001Google Scholar
  6. 6.
    McGaughey, W. H., Insect resistance to the biological insecticide Bacillus thuringiensis, Science, 1985, 229: 193–195.PubMedCrossRefGoogle Scholar
  7. 7.
    Tabashnik, B. E., Cushing, N. L., Finson, N. et al., Field development of resistance to Bacillus thuringiensis in diamonback moth (Lepidoptera: Plutellidae), J. Econ. Entomol., 1990, 83: 1671–1676Google Scholar
  8. 8.
    Liang, G. M., Tan, W. J., Guo, Y. Y., Study on screening and inheritance mode of resistance to Bt transgenic cotton in cotton bollworm, Acta Entomologica Sinica (in Chinese), 2000, 43(Suppl.): 57–62.Google Scholar
  9. 9.
    Liang, G. M., Tan, W. J., Guo, Y. Y., Study on the resistance screening and cross-resistance of Cotton Bollworm to Bacillus thuringiensis (Berliner), Scientia Agricultura Sinica (in Chinese), 2000, 33(4): 46–53.Google Scholar
  10. 10.
    Ferré, J., Van Rie, J., Biochemistry and genetics of insect resistance to Bacillus thuringiensis, Annu. Rev. Entomol., 2002, 47: 501–533.PubMedCrossRefGoogle Scholar
  11. 11.
    Vadlamudi, R. K., Ji, T. H., Bulla, L. A. Jr., A specific Binding Protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. Berliner, J. Biol. Chem., 1993, 268: 12334–12340.PubMedGoogle Scholar
  12. 12.
    Vadlamudi, R. K., Weber, E., Ji, I. et al., Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis, J. Biol. Chem., 1995, 270: 5490–5494.PubMedCrossRefGoogle Scholar
  13. 13.
    Takeichi, M., Cadherin cell adhesion receptors as a morphogenetic regulator, Science, 1991, 251: 1451–1455.PubMedCrossRefGoogle Scholar
  14. 14.
    Takeichi, M., Morphogenetic roles of classic cadherins, Curr. Opin. Cell Biol., 1995, 7: 619–627.PubMedCrossRefGoogle Scholar
  15. 15.
    Vleminckx, K., Vakaet, L., Jr Mareel, M. et al., Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role, Cell, 1991, 66: 107–119.PubMedCrossRefGoogle Scholar
  16. 16.
    Dorsch, J. A., Candas, M., Griko, N. B. et al., Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca Sexta: Involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol., 2002, 32: 1025–1036.PubMedCrossRefGoogle Scholar
  17. 17.
    Hara, H., Atsumi, S., Yaoi, K. et al., A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae, FEBS Lett., 2003, 538(1–3): 29–34.PubMedCrossRefGoogle Scholar
  18. 18.
    Nagamatsu, Y., Toda, S., Yamaguchi, F. et al., Identification of Bombyx mori midgut receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin, Biosci. Biotechnol. Biochem., 1998, 62(4): 718–726.PubMedCrossRefGoogle Scholar
  19. 19.
    Gahan, L. J., Gould, F., Heckel, D. G., Identification of a gene associated with Bt resistance in Heliothis virescenes, Science, 2001, 293: 857–860.PubMedCrossRefGoogle Scholar
  20. 20.
    Morin, S., Biggs, R. W., Sisterson, M. S. et al., Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm, PNAS, 2003, 100(9): 5004–5009.PubMedCrossRefGoogle Scholar
  21. 21.
    Liang, G. M., Tan, W. J., Guo, Y. Y., An improvement in the technique of artificial raring cotton bollworm, Plant Protection (in Chinese), 1999, 25: 15–17.Google Scholar
  22. 22.
    Luo, K., Lu, Y. J., Adang, M. J., A 106-kDa form of aminopeptidase is a receptor for Bacillus thuringiensis Cry1C deltaen-dotoxin in the brush border membrane of Manduca Sexta, Insect Biochem. Mol. Biol., 1996, 26: 33–40.CrossRefGoogle Scholar
  23. 23.
    Wang, G. R., Liang, G. M., Wu, K. M. et al., Cloning and sequencing of a gene encoding aminopeptidase N in the mid-gut of Helicoverpa armigera (Hübner), Agricultural Sciences in China, 2003, 2(7): 760–767.Google Scholar
  24. 24.
    Sambrook, K. J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory, 1989.Google Scholar
  25. 25.
    Pagni, M., Iseli, C., Junier, T. et al., TrEST, trGEN and Hits: Access to databases of predicted protein sequences, Nucleic Acids Research, 2001, 29: 148–151.PubMedCrossRefGoogle Scholar
  26. 26.
    Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites, Protein Engineering, 1997, 10: 1–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Wang, G. R., Wu, K. M., Guo, Y. Y., Cloning, expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner), Insect Biochem. Mol. Biol., 2003, 33: 115–124.PubMedCrossRefGoogle Scholar
  28. 28.
    Cao, W. H., Dong, Y., Zhang, J. S., Chen, S. Y., Characterization of an ethylene receptor homolog gene from rice, Science in China, Ser. C, 2003, 46 (4): 370–378.CrossRefGoogle Scholar
  29. 29.
    Hofte, H., Whigeley, H. R., Insecticidal crystal proteins of Bacillus thuringiensis, Microbiol. Rev., 1989, 53: 242–255.PubMedGoogle Scholar
  30. 30.
    English, L., Slatin, S. L., Mode of action of delta-endotoxins from Bacillus thuringiensis: A comparison with other bacterial toxins, Insect Biochem. Mol. Biol., 1992, 22(1): 1–7.CrossRefGoogle Scholar
  31. 31.
    Gill, S. S., Cowles, E. A., Pietrantonio, P. V., The mode of action of Bacillus thuringiensis endotoxins, Annu. Rev. Entomol., 1992, 37: 615–636.PubMedCrossRefGoogle Scholar
  32. 32.
    Knight, P. J., Knowles, B. H., Ellar, D. J., Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin, J. Biol. Chem., 1995, 28: 270(30): 17765–17770.PubMedCrossRefGoogle Scholar
  33. 33.
    Garner, K. J., Hiremath, S., Lehtoma, K., Valaitis, A. P., Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Actoxin, Insect Biochem. Mol. Biol., 1999, 29(6): 527–535.PubMedCrossRefGoogle Scholar
  34. 34.
    Wu, K., Guo, Y., Geographic variation in susceptibility of Helicoverpa armigera (Lepidoptra: Noctuidae) to Bt insecticidal protein in China, Journal of Economic Entomology, 1999, 92(2): 273–278.Google Scholar
  35. 35.
    Wu, K., Guo, Y., Lv, N. et al., Resistance monitoring of Helicoverpa armigera (Lepidoptera: Noctuidae) to Bt insecticidal protein in China, Journal of Economic Entomology, 2002, 95(4): 826–831.PubMedCrossRefGoogle Scholar
  36. 36.
    Environmental Protection Agency, The Environmental Protection Agency’ White Paper on Bt Plant-Pesticide Resistance Management, Washington: EPA Publication, 1998, 739-S-98-001.Google Scholar

Copyright information

© Science in China Press 2005

Authors and Affiliations

  • Guirong Wang
    • 1
  • Kongming Wu
    • 1
  • Gemei Liang
    • 1
  • Yuyuan Guo
    • 1
  1. 1.State Key Laboratory of Plant Disease and Insect Pests, Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina

Personalised recommendations