Cloning And Nucleotide Sequencing of A Nematicidal Gene From Bacillqs thuringiensis subsp. aegypti

Abstract

Bacillus thuringiensis subsp. aegypti is a novel isolate charactrized by its wide host range and its pesticidal activity. its insecticidal activity was previously studied against lepidopteran, dipteran and coleopteran insects, but recently it was found to have strong nematocidal activity. This activity was associated with vegetative cells and spores, in both culture supernatant and cell pellets. The main objectives of the present investigation were: (i) The determination and partial purification of the nematocidal proteins isolated from either the bacterium or its supernatant, and; (ii) Cloning the gene that encodes for the nematocidal protein(s).

(i) The determination and purification of the nematocidal protein of Bacillus thuringiensis were carried out by fractionating the vegetative cells and spores with ammonium sulfate. Before fractionation, each supernatant was divided into two parts, one is boiled, to eliminate any heat labile toxins, and the other part was left without boiling. The 60% and 40% fractions obtained from Bacillus thuringiensis vegetative cells and spores were further subjected to bioassays against the nematode larvae. The bioassay