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, 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.
Highlights
The Bacillus thuringiensis (Bt) has been used as a successful biological insecticide for more than 40 years and is a uniquely specific, safe, and effective tool for the control of a wide variety of insect pests [1]
The present study describes screening of indigenous isolates of Bt for cry1A genes by Polymerase Chain Reaction (PCR)-Restriction fragment length polymorphic (RFLP) and cloning of a new truncated cry1Ac gene from an Indian isolate of Bt
Based on PCR-RFLP data, the Bt isolate, T15 which showed 100 per cent mortality in H. armigera and having cry1Ac gene alone was selected for cloning of DNA fragments of ~2.1 kb corresponding to 3’-truncated cry1A gene
Summary
The Bt has been used as a successful biological insecticide for more than 40 years and is a uniquely specific, safe, and effective tool for the control of a wide variety of insect pests [1]. Continuous exposure of a single Bt protein in Bt cotton can lead to resistance development in lepidopteran insects [5,6]. Cry1Ac protein is about 30 fold less toxic to H. armigera than to Heliothis virescens, the original target pest of transgenic cotton in USA [7]. A large number of Bt strains have been isolated and many types of insecticidal crystal proteins genes have been cloned. New variants of the already known cry gene subgroups could encode crystal proteins with significant difference in the level and spectrum of toxicity due to variation in OPEN ACCESS. The present study describes screening of indigenous isolates of Bt for cry1A genes by PCR-RFLP and cloning of a new truncated cry1Ac gene from an Indian isolate of Bt
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