Cloning a mosquitocidal fragment of Bacillus thuringiensis subsp. israelensis and location of the insect binding specificity domain of the 130 kDa toxin gene

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Robinson, Mary J.
Vann, Carolyn N.
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Thesis (M.S.)
Department of Biology
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Various strains of Bacillus thuringiensis Mt.) produce crystalline endotoxins specific for larvae of different insect classes. Two strains, B.t. subspp. israelensis and kurstaki produce similar 130 kDa toxins encoded by the CryIVB gene (toxic to Diptera) and the CryIA gene (toxic to Lepidoptera), respectively. The N-terminal region of the CryIVB gene was cloned into the Escherichia coli expression vector pKX223-3. A mosquitocidal transformant was obtained as determined by mosquito bioassays. The gene fragment, if stable, can be cloned into cyanobacteria to achieve biological control of mosquito-borne diseases. A second goal was to identify the binding specificity domain of the CryIVB gene which encodes the portion of the protein toxin that binds the insect midgut causing cell lysis and death. Two potential insect binding specificity domains identified by computer analyses were switched with a known binding specificity region of the CryIA gene. The polymerase chain reaction was utilized to obtain gene fragments of the CryIVB gene which replaced the CryIA gene binding specificity domain. The resulting recombinant clones carrying the CryIA gene containing the .000nd proposed insect binding specificity domain of the CryIVB gene were fotsd to be mosquitocidal.