| Summary: | 碩士 === 淡江大學 === 生命科學研究所碩士班 === 98 === The availability of effective and cheap insecticides heralded an agricultural revolution. The insecticides not only help farmers increase yields, but also lower food prices. However, use of these insecticides is detrimental since some of these persist in the environment and accumulate in living organisms, causing various fatal diseases and are also toxic to nontarget species. In time new resistant strains of insects emerge, requiring increased doses of insecticides and introduction of new insecticides. Recently, bioinsecticides are being used as an alternative to the insecticides. Bioinsecticides are certain types of pesticides derived from natural materials including animals, plants, and bacteria. The toxic action of bioinsecticides is often specific to a single group or species of insects, does not hazardous to non-target species. They are also degraded in sunlight. The most widely used bioinsecticides are microbial pesticides.
Pseudomonas taiwanensis TKU015 was isolated from Taiwan soil and was found that the amino acid sequence of one of its insecticidal proteins was similar to Pseudomonas entomphila L48, so the primers was designed to clone this insecticidal protein gene in further studies. By using polymerase chain reaction method, the 2-kb DNA fragments encoding insecticidal toxin was obtained as described previously. By using Genome WalkerTM kit, the 3-kb fragments encoding full-length insecticidal toxin was obtained. The insecticidal toxin genes were subcloned into the pET-32 Xa/Lic vector and then transformed into E. coli BL21. The recombinant insecticidal toxins were expressed by the transformed E. coli BL21 by induction with IPTG at 37 degrees overnight. The target proteins fused with His-tag were purified and de-salted. Finally, the purified insecticidal proteins were analyzed its insecticidal effect on Drosophila larvae.
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