Cloning of a New Truncated <i>cry</i>1<i>Ac</i> Gene from an Indian Isolate of <i>Bacillus thuringiensis</i>

Abstract

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, Cry 1 A toxins are important because of high toxicity to lepidopteran pests and their widespread distribution among Bt strains. In Cry 1 A 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 cry 1 A gene(s) corresponding to toxin fragment. RFLP analysis of cry 1 A amplicons obtained from the four isolates of Bt showed presence of cry 1 Ac -type gene alone in three isolates. One of the cry 1 Ac -postive isolate s , T15 which showed 100 percent mortality in Helicoverpa armigera , was selected for cloning of DNA fragment of about 2.1 kb containing 3’-truncated cry 1 Ac gene. Nucleotide sequence data generated for 3’-truncated cry 1 Ac gene of T15 showed 98 to 99 percent homology with 1958 bp of already reported sequences of all cry 1 Ac genes ( cry 1 Ac 1 to cry 1 Ac 24). Deduced amino acid sequence of cry 1 Ac of Bt strain, T15 showed one to four percent variation in comparison to all reported Cry 1 Ac holotypes ( Cry 1 Ac 1 to Cry 1 Ac 24) by differing at 5 to 19 positions. This suggests that the cry 1 Ac toxin of Bt isolate, T15 is a new kind of its group.