M.Sc. Tezi Görüntüleme | |||||||||||||||||||||
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| Summary: Bacillus thuringiensis, is a soil bacterium which produce insecticidal crystal toxin against some insect orders. Because of this feature, Cry3Aa (δ-endotoksin) proteins have great importance in terms of biologicalcontrol. In the present study, cloning in a different host Escherichia coli, characterization, expression and bioassay of cry3Aa gene of Bacillus thuringiensis (Bt Xd3) isolated from Xyleborus dispar which is a hazelnut pest were performed. For this reason cry3Aa gene was amplified from Bt Xd3 bacteria by PCR. For identifying the nucleotide sequence of the amplified gene, PCR product was cloned into the pGEM-T Easy cloning vector. Nucleotide sequence of cry3Aa gene and its deduced amino acid sequence were determined and compared with other cry3Aa genes. The Cry3Aa protein belong to B.thuringiensis (Bt Xd3) has %100 homology with Cry3Aa2, Cry3Aa3, Cry3Aa5 and Cry3Aa6 proteins of Bacillus thuringiensis subsp tenebrionis (morrisoni) in literature. Bt Xd3 cry3Aa gene sequence was recorded in gene bank as cry3Aa12. To express the protein produced by cry3Aa gene in a different host and detect its insecticidal activity the gene was cloned into pET-28a(+) expression vector. The protein expressed in expression vector was isolated and SDS-PAGE analysis of the protein was performed. 73kDa protein band belonged to cry3Aa gene was observed on polyacrylamide gel. Insecticidal activity of this obtained Cry3Aa δ-endotoxin against Alphitobius diaperinus larvae belong to Coleoptera order was determined. As a result of the performed bioassay, recombinant protein expressed in E.coli BL21(DE3) is found as less effective than spor-crystal complex in B. thuringiensis on Coleoptera larvae.KeyWords: Bacillus thuringiensis, Crystal Protein, Cry3Aa δ-endotoxin and Expression | |||||||||||||||||||||