Master Degree / Yüksek Lisans Tezleri
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Master Thesis Isolation and Characterization of Bacillus Thuringiensis Strains From Different Grain Habitats(Izmir Institute of Technology, 2004) Apaydın, Özgür; Güneş, HaticeBacillus thuringiensis is a Gram positive, facultative anaerob bacteria that produces proteins toxic against different insect species. This feature makes it the most widely used biological control agent in agriculture. Since B. thuringiensis strains have great genetic diversity, the toxic behaviours of these strains differ from region to region. Native B. thuringiensis strains are isolated from different habitats and characterized to determine their toxic potential all over the world. The aim of this study was to isolate B. thuringiensis strains from different grain habitats in Central Anatolia and Aegean Regions, and to investigate their phenotypic and genotypic characterizations. Total 96 samples containing soil, grain, stored product dust, straw and various residues were collected from wheat farms, grain silos, haylofts and caves in Ereli/Konya, Takale/Karaman, Nikfer/Denizli, and Bozbük/Söke under aseptic conditions. Seven hundred bacteria were isolated from these samples by sodium acetate selection and heat treatment. For phenotypic characterization, 500 of these isolates were grown for 48 h and crystal protein production was observed by phase contrast microscobe during spore formation. One hundred and sixty three of the bacterial colonies were identified as B. thuringiensis. The isolates were divided into 5 different groups based on the shape of the crystals that they produced. Spherical type crystal morphology was mostly observed type among the others. For genotypic characterization, the cry gene content of the isolates were screened by polymerase chain reaction (PCR) analysis. In addition, chromosomal DNA analysis of 34 isolates by Pulsed Field Gel Electrophoresis (PFGE) as well as plasmid DNA profiling for all isolates were also carried out. One hundred and three isolates were positive for 5 different cry genes (cry1, cry2, cry3, cry4, cry9) examined by PCR. Among all cry genes examined, cry1 and cry9 genes were mostly found in the isolates. Morover, plasmid profiling of the isolates indicated that a 15 kb DNA band was present in all the isolates; however, some of them had more than one DNA band at different sizes. Finally, chromosomal DNA profiling by PFGE showed different DNA patterns for isolates containing the same cry gene which suggest a high level of diversity among the B. thuringiensis strains isolated. Further studies related with extensive genetic characterization and toxic activity of each B. thuringiensis strain will give more comprehensive results on biodiversity of B. thuringiensis strains in Anatolia.Master Thesis Isolation of Bacillus Thuringiensis and Investigation of Crystal Protein Genes(Izmir Institute of Technology, 2002) Çetinkaya, Fatoş Tuba; Güneş, HaticeBacillus thuringiensis is a ubiquitous, gram-positive and spore-forming bacterium. During sporulation, it produces intracellular crystal proteins (cry proteins), which are toxic to insects. Because of its insecticidal activity, it has been used for nearly fifty years to control certain insect species among the orders Lepidoptera, Coloeptera, and Diptera. However, it is still necessary to search for more toxins to control other insect orders and to provide alternatives for coping with the problem of insect resistance. The genetic diversity of B. thuringiensis strains shows differences according to the regions where they were isolated. Thus, each habitat may contain novel B. thuringiensis strains, which have some toxic effects on target spectra of insects. The aim of this study was to isolate B. thuringiensis strains from different environments and to identify the crystalline protein gene content of the isolates. Sixty five samples including soil, stored product dust, insect cadavers, and dry leaf residues were collected from Akhisar/Manisa, İzmir, and Ereğli/Konya. Three approaches were applied for the isolation of B. thuringiensis: sodium acetate selection, heat treatment, and endospore staining. Polymerase Chain Reaction (PCR) method was used for the characterization of cry gene content of B. thuringiensis strains. The universal primers specific to cry 1, cry2, cry 3, and cry 9 genes were used to detect the type of cry gene carried by each environmental isolate of B. thuringiensis strains. In addition, 16S rRNA based PCR-restriction fragment length polymorphism (RFLP) was carried out to confirm B. thuringiensis strains. Finally, SDS-PAGE analysis was optimized to detect protein profiles of crystal proteins obtained from B. thuringiensis isolates. It was found that, 136 of 359 isolates showed B. thuringiensis-like colony morphology and subterminal endospore position. One hundred isolates were screened by PCR and 18 of them were found to contain cry genes (5 cry 1, 3 cry3, and 10 cry 9). However, the cry 2 gene was not detected from any isolates. 16S rRNA based PCR-RFLPfor 18 isolates gave the same restriction pattern as positive controls, indicating that all 18 isolates were B. thuringiensis. SDS-PAGE studies for Cry 9 proteins of the isolates exhibited different protein profile from positive control of B thuringiensis strain.