Cloning of a New Truncated cry1Ac Gene from an Indian Isolate of Bacillus thuringiensis


Transgenic Bt crops producing insecticidal crystal proteins from Bacillus thuringiensis (Bt), so-called Cry toxins, have proved useful in controlling insect pests. Among the cry toxins, Cry1A toxins are important because of high toxicity to lepidopteran pests and their widespread distribution among Bt strains. In Cry1A proteins, toxin fragment is comprised of about 620 amino acids of N-terminal region and C-terminal half is not required for toxicity. Four indigenous isolates of Bt viz., T15, T16, T20 and T31 were screened by PCR-RFLP for 3’-truncated cry1A gene(s) corresponding to toxin fragment. RFLP analysis of cry1A amplicons obtained from the four isolates of Bt showed presence of cry1Ac-type gene alone in three isolates. One of the cry1Ac-postive isolates, T15 which showed 100 percent mortality in Helicoverpa armigera, was selected for cloning of DNA fragment of about 2.1 kb containing 3’-truncated cry1Ac gene. Nucleotide sequence data generated for 3’-truncated cry1Ac gene of T15 showed 98 to 99 percent homology with 1958 bp of already reported sequences of all cry1Ac genes (cry1Ac1 to cry1Ac24). Deduced amino acid sequence of cry1Ac of Bt strain, T15 showed one to four percent variation in comparison to all reported Cry1Ac holotypes (Cry1Ac1 to Cry1Ac24) by differing at 5 to 19 positions. This suggests that the cry1Ac toxin of Bt isolate, T15 is a new kind of its group.

Share and Cite:

A. Ramalakshmi, R. Manikandan, V. Balasubramani and V. Udayasuriyan, "Cloning of a New Truncated cry1Ac Gene from an Indian Isolate of Bacillus thuringiensis," Advances in Microbiology, Vol. 4 No. 1, 2014, pp. 55-62. doi: 10.4236/aim.2014.41009.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] E. W. Nester, L. S. Thomashow, M. Metz and M. Gordon, “100 Years of Bacillus thuringiensis Bacillus thuringiensis: A Critical Scientific Assessment. American Society for Microbiology,” Washington DC, 2002.
[2] E. Schnepf and H. R. Whiteley, “Cloning and Expression of the B. thuringiensis Protein Gene in E. coli,” Proceedings of the National Academy of Sciences, Vol. 78, No. 5, 1981, pp. 2893-2897.
[3] A. Bravo, S. Sarabia, L. Lopez, H. Ontiveros, C. Abarca, A. Ortiz, M. Ortiz, L. Lina, F. J. Villalobos, G. Pena, M. E. Nunez-Valdez, M. Soberon and R. Quintero, “Characterization of Cry Genes in a Mexican B. thuringiensis Strain Collection,” Applied and Environmental Microbiology, Vol. 64, 1998, pp. 4965-4972.
[4] D. Uribe, W. Masrtinez and J. Ceron, “Distribution and Diversity of Cry Genes in Native Strains of B. thuringiensis Obtained from Different Ecosystems from Colombia,” Journal of Invertebrate Pathology, Vol. 82, No. 2, 2003, pp. 119-127.
[5] R. J. Akhurst, W. James, L. Bird and C. Beard, “Resistance to the Cry1Ac-Endotoxin of B. thuringiensis in the Cotton Bollworm, H. armigera (Lepidoptera: Noctuidae),” Journal of Economic Entomology, Vol. 96, No. 4, 2003, pp. 1290-1299.
[6] B. E. Tabashnik, “Evolution of Resistance to B. thuringiensis,” Annual Review of Entomology, Vol. 39, 1994, pp. 47-79.
[7] C. Liao, D. G. Heckel and R. Akhurst, “Toxicity of B. thuringiensis Insecticidal Protein for Helicoverpa armigera and H. punctigera (Lepidoptera: Noctuidae), Major Pests of Cotton,” Journal of Invertebrate Pathology, Vol. 80, No. 1, 2002, pp. 55-63.
[8] J. Xue, G. M. Liang, N. Crickmore, H. Li, K. He, F. Song, X. Feng, D. Huang and J. Zhang, “Cloning and Characterization of a Novel Cry1A Toxin from Bacillus thuringiensis with High Toxicity to the Asian Corn Borer and Other Lepidopteran Insects,” Microbiology Letters, Vol. 280, No. 1, 2008, pp. 95-101.
[9] V. Udayasuriyan, A. Nakamura, A. Mori, H. Masaki and T. Uozumi, “Cloning of a New crylA(a), Gene from B. thuringiensis Strain FU-2-7 and Analysis of Chimeric cry1A(a) Proteins of Toxicity,” Bioscience, Biotechnology, and Biochemistry, Vol. 58, No. 5, 1994, pp. 830-835.
[10] H. Fujimoto, K. Itoh, M. Yamamoto, J. Kyozuka and K. Shimamoto, “Insect Resistant Rice Generated by Introduction of a Modified δ-Endotoxin Gene of Bacillus thuringiensis,” Biotechnology, Vol. 11, 1993, pp. 1151-1155.
[11] S. Kalman, K. L. Kiehne, N. Cooper, M. S. Reynoso and T. Yamamoto, “Enhanced Production of Insecticidal Proteins in Bacillus thuringiensis Strains Carrying an Additional Crystal Protein Gene in the Chromosomes,” Applied and Environmental Microbiology, Vol. 61, 1995, pp. 3063-3068.
[12] J. Sambrook, E. F. Fritsch and T. M. Maniatis, “Molecular Cloning: A Laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
[13] T. A. Hall, “BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis,” 1999.
[14] E. Ben-Dov, A. Zaritsky, E. Dahan, Z. Barak, R. Sinai, R. Manasherob, A. Khamraev, E. Troitskaya, A. Dubitsky, N. Berezina and Y. Margalith, “Extended Screening by PCR for Seven cry Group Genes from Field-Collected Strains of B. thuringiensis,” Applied and Environmental Microbiology, Vol. 63, 1997, pp. 4883-4890.
[15] W. S. Kuo and K. F. Chak, “Identification of Novel cryType Genes from B. thuringiensis Strains on the Basis of Restriction Fragment Length Polymorphism of the PCRAmplified DNA,” Applied and Environmental Microbiology, Vol. 62, 1996, pp. 1367-1377.
[16] J. Wang, A. Boets, J. van Rie and G. Ren, “Characterization of cry1, cry2 and cry9 Genes in B. thuringiensis Isolates from China,” Journal of Invertebrate Pathology, Vol. 82, No. 1, 2003, pp. 63-71.
[17] F. Song, J. Zhange, A. Gu, Y. We, L. Han, K. He, Z. Chan, J. Yao, Y. Hu, G. Li and D. Huang, “Identification of cry1I-Type Genes from B. thuringiensis Strains and Characterization of a nOvel cry1I Type Gene,” Applied and Environmental Microbiology, Vol. 69, No. 9, 2003, pp. 5207-5211.
[18] H. Hofte and H. R. Whiteley, “Insecticidal Crystal Proteins of B. thuringiensis,” Microbiology Reviews, Vol. 53, 1989, pp. 242-255.
[19] H. W. Park, D. K. Bideshi and B. A. Federici, “Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesis in Bacillus thuringiensis to Improve Stability and Yield,” Applied and Environmental Microbiology, Vol. 66, No. 10, 2000, pp. 4449-4455.
[20] E. Schnepf, N. Crickmore, J. Van Rie, D. Lerecurs, J. Baum, J. Feitelson, J. D. R. Zeigler and D. H. Dean, “B. thuringiensis and Its Pesticidal Crystal Proteins,” Microbiology and Molecular Biology Reviews, Vol. 62, 1998, pp. 775-806.
[21] M. K. Lee, T. H. You, A. Curtiss and D. H. Dean, “Involvement of Two Amino Acid Residues in the Loop Region of Bacillus thuringiensis Cry1Ab Toxin in Toxicity and Binding to Lymantria dispar,” Biochemical and Biophysical Research Communications, Vol. 229, No. 1, 1996, pp. 139-146.
[22] P. Grochulski, L. Masson, S. Borisova, M. Pusztai-Carey, J. L. Schwartz, R. Brousseau and M. Cygler, “B. thuringiensis CryIA(a) Insecticidal Toxin: Crystal Structure and Channel Formation,” Journal of Molecular Biology, Vol. 254, No. 3, 1995, pp. 447-464.
[23] E. Gazit, P. L. Rocca, M. S. P. Sansom and Y. Shai, “The Structure and Organization within the Membrane of the Helices Composing the Pore-Forming Domain of Bacillus thuringiensis Delta-Endotoxin Are Consistent with an ‘Umbrella-Like’ Structure of the Pore,” Proceedings of the National Academy of Sciences, Vol. 95, No. 21, 1998, pp. 12289-12294.

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.