CLONING, EXPRESSION AND TOXICITY OF A MOSQUITOCIDAL TOXIN GENE OF BACILLUS-THURINGIENSIS SUBSP MEDELLIN

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces paraspora l crystalline inclusions which are toxic to mosquito larvae. It has be en shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained . The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragmen t into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estima ted by using first instar laboratory reared Aedes aegypti, and Culex q uinquefasciatus larvae challenged with whole crystals. Toxicity, resul ts indicate that the purified inclusions from the recombinant Bti stra in were toxic to all mosquito species tested, although the toxicity wa s not as high as the one produced by the crystal of the Btmed wild typ e strain. Poliacrylamide gel electrophoresis indicate that the inclusi ons produced by the recombinant strain Bti (pBTM3) were mainly compose d of the 94 kDa protein of Btmed, as it was determined by Western blot .