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Cloning, expression and toxicity of a mosquitocidal toxin gene of Bacillus thuringiensis subsp. medellin
N Restrepo1, D Gutierrez, M M Patiño
1Unidad de Biotecnologia y Control Biológico, Corporación para Investigaciones Biológicas, Medellín, Colombia.
Abstract:
Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been 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 polyclonal 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 fragment 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 estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Polyacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot.
Insights
Bacillus thuringiensis subsp. medellin (Btmed) genes were cloned and expressed in a crystal-negative strain of Bacillus thuringiensis subsp. israelensis (Bti). The recombinant Bti produced toxic inclusions effective against mosquito larvae.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Insect Toxicology
Background:
- Bacillus thuringiensis (Bt) subsp. medellin produces insecticidal crystalline inclusions toxic to mosquito larvae.
- These inclusions are primarily composed of 94, 68, and 28-30 kDa proteins.
Purpose of the Study:
- To clone and express the gene encoding the 94 kDa crystal protein of Btmed in a heterologous Bti strain.
- To assess the toxicity of the recombinant Bti strain against mosquito larvae.
Main Methods:
- Cloning of Btmed DNA using Lambda Zap II kit and screening with antibodies against the 94 kDa protein.
- Subcloning the 94 kDa gene into a shuttle vector and transforming a crystal-negative Bti strain (4Q2-81).
- Assessing larval toxicity using Aedes aegypti and Culex quinquefasciatus, and analyzing inclusion proteins via SDS-PAGE and Western blot.
Main Results:
- The 94 kDa toxin gene from Btmed was successfully cloned into a 4.4 kb DNA fragment.
- Recombinant Bti strain (pBTM3) produced inclusions mainly composed of the 94 kDa Btmed protein.
- Inclusions from recombinant Bti showed toxicity against Aedes aegypti and Culex quinquefasciatus larvae, though less potent than wild-type Btmed.
Conclusions:
- The 94 kDa protein gene from Btmed is functional when expressed in Bti.
- Recombinant Bti strains expressing Btmed toxins offer potential for mosquito control strategies.
- Further optimization may enhance the potency of recombinant Bti for improved larvicidal activity.