Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringi
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RESEARCH
Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis V. Prathap Reddy • N. Narasimha Rao • P. S. Vimala Devi • S. Sivaramakrishnan M. Lakshmi Narasu • V. Dinesh Kumar
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Published online: 9 December 2012 Ó Springer Science+Business Media New York 2012
Abstract A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as
cry1Ab26 by the B. thuringiensis d-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(?) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.
Electronic supplementary material The online version of this article (doi:10.1007/s12033-012-9627-3) contains supplementary material, which is available to authorized users.
Introduction
V. P. Reddy N. N. Rao P. S. V. Devi V. D. Kumar (&) Directorate of Oilseeds Research, ICAR, Rajendranagar, Hyderabad 500030, India e-mail: [email protected]; [email protected] V. P. Reddy e-mail: [email protected] N. N. Rao e-mail: [email protected] P. S. V. Devi e-mail: [email protected] S. Sivaramakrishnan Institute of Biotechnology, Acharya N. G. Ranga Agricultural University, Hyderabad 500030, India e-mail: [email protected] M. L. Narasu Department of Biotechnology, JNTU, Kukatpally, Hyderabad 500080, India e-mail: [email protected]
Keywords Bacillus thuringiensis Bioassay Cloning Protein expression Cry1ab26 ELISA SDS-PAGE
Bacillus thuringiensis (Bt) is a gram-positive, aerobic bacterium that has been used as a successful biologic insecticide. The insecticidal property of Bt is due to the production of insecticidal crystal proteins (ICPs) also called d-endotoxins, consisting of one or more crystal (Cry) proteins and Cytolitic (Cyt) toxins [1]. The cry genes have been categorized according to the class of insects they are effective against [2]. Bt strains carry different classes of cry genes [3], and therefore the overall toxicity profile of the isolate depends on regulation of expression of individual cry genes, the relative amounts of the various protoxins included, solubility and their proteolytic processing by different proteases within larval mi
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