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Chimeric δ-endotoxins of Bacillus thuringiensis with increased activity against Helicoverpa armigera

Published online by Cambridge University Press:  01 June 2011

Anuradha Chelliah ,
Gorakh Prasad Gupta ,
Sasikumar Karuppiah  and
Polumetla Ananda Kumar
Anuradha Chelliah*
Affiliation:
National Research Centre for Banana, Thogamalai Road, Thayanur Post, Tiruchirapalli620 102, Tamil Nadu, India
Gorakh Prasad Gupta
Affiliation:
Division of Entomology, Indian Agricultural Research Institute, New Delhi110 012, India
Sasikumar Karuppiah
Affiliation:
H.H. Raja's College, Pudukottai622 001, Tamil Nadu, India
Polumetla Ananda Kumar
Affiliation:
National Research Centre on Plant Biotechnology, New Delhi110 012, India
*
*E-mail: anuradha.chelliah@gmail.com
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Abstract

We selected 50 crystal (Cry) toxins specific to lepidopteran insects. Phylogenetic relationships of whole toxins as well as individual domains revealed that Cry1Jb and Cry1Ac are distantly placed but related to reduced cross-resistance in Helicoverpa armigera. Multiple alignments of Cry1Jb and Cry1Ac amino acid sequences showed significant differences in the composition and length of the loops of domains II and III. This was further confirmed by the superpositioning of 3D structures. cry1Jb and cry1Ac genes cloned in expression vectors were overexpressed in Escherichia coli Castellani & Chalmers, and proteins were harvested. Insect bioassays revealed that the wild-type Cry1Jb and Cry1Ac proteins showed differential specificity to H. armigera (Hübner), Spodoptera litura (Fab.) and Earias vittella F. Chimeric genes were constructed by exchanging the domains between cry1Jb and cry1Ac and overexpressed in BL21(DE3). Substitution of domain III of Cry1Jb with that of Cry1Ac enhanced its activity against H. armigera 7.8-fold. Bioassay of the parental and chimeric toxins against S. litura revealed no activity even at the discriminative dose of 100 μg/ml, but a significant difference in growth was observed. The present results, along with previous domain swapping experiments, suggest that protein engineering not only reveals the mechanism by which endotoxins work, but also generates novel toxins with enhanced toxicity/broader specificity.

Keywords

chimeric toxinsprotein engineeringdomain swappingCry1JbCry1AcLC50Helicoverpa armigeraSpodoptera lituraEarias vittella
Type
Research Paper
Information
International Journal of Tropical Insect Science , Volume 31 , Issue 1-2 , June 2011 , pp. 59 - 68
Copyright
Copyright © ICIPE 2011

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