Cloning and analysis sequence of Bacillus thuringiensis ENTOMOCIDUS INA 288

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Research Paper 01/08/2019
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Cloning and analysis sequence of Bacillus thuringiensis ENTOMOCIDUS INA 288

Akhmad Rizali
J. Bio. Env. Sci.15( 2), 7-12, August 2019.
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B. thuringiensis has been studied worldwide over the past decades, mainly because this gram-positive bacterium produces significant amounts of crystal protein toxic activity against economically important insect larvae. Most strains of B. thuringiensis produce delta-endotoxin crystals toxic to lepidopteran insects such moth. But some strains of B. thuringiensis produce delta-endotoxin crystals toxic to dipteran insects such as mosquitoes and blackflies. Numerous chemical insecticides have been used to control some insects. While chemical insecticides have knock down effect, they are too expensive in the developing countries and harmful to both humans and the environment. In addition, target insect pests develop biological resistance rapidly especially at higher rates of application. Thus, the increase in pesticidal application to control this pest has urged to researcher to search for biological control alternatives that would be a good component of Integrated Pest Management. To clone and analyze B. thuringiensis gene fragments isolated by restriction digest PCR. Specific primers were designated to amplify the genes of B. thuringiensis serovar entomocidus INA 288, and the PCR products were classified and re-amplyfied by PCR to obtained the fragments for subsequent purification and cloning into the pGEM-T vectors, followed by rapid identification. The recombinant plasmid were extracted from positive clones and the target gene fragments were sequenced. The sequence analysis showed that cry4Aa had similarity with cryINA 288. It seems a novel mosquitocidal cry gene. It may be concluded that dipteracidal polypeptide of entomocidus INA 288 (cryINA 288) consisted of 703 amino acids with conserved blocks and amino acid sequence was aligned to that of cry4Aa. Furthemore, an aligment of the conserved block sequences among the cry INA 288 and other mosquitocidal proteins was performed.


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