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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)

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20051
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Subject: Agricultural Biotechnology × Thesis Type: M.Sc × Author: AMARENDRA, J ×
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    ThesisItemOpen Access
    Molecular Characterization of Bacillus thuringiensis Isolates
    (ACHARYA N.G.RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD, 2005) AMARENDRA, J; Sokka Reddy, S
    Bacillus thuringiensis (Bt) is a gram-positive, spore producing, soil bacterium. Various strains of Bacillus thuringiensis (Berliner, 1911) produce different insecticidal crystal proteins which are encoded by cry genes. The present investigation was undertaken with the aim of characterizing 120 local B. thuringiensis isolates with respect to the subspecies to which they might belong and also to identify the cry genes present in these 120 isolates, which were collected and maintained at the Directorate of Oilseeds Research, Hyderabad, along with 23 standard isolates belonging to 11 subspecies of B. thuringiensis obtained from the Bacillus Genetic Stock Centre, Ohio, USA and 3 commercial formulations of B. thuringiensis, were used for molecular characterization in the present study. Initially rep-PCR analysis was done with BOX, Enterobacterial Repetitive Intergenic Consensus sequence (ERIC) and Repetitive Extragenic Palindromic sequences (REP) primers. From the rep-PCR analysis, a dendrogram was constructed which revealed that most of the isolates had higher genetic relatedness to Bacillus thuringienis subspecies kurstaki. Finally, cry gene profiling of all the 120 isolates was done by both universal and gene specific primers for cry genes. When universal primers were used, it was found that majority of the isolates carried cry1 and cry2 genes, indicating their effectiveness against lepidopteran pests. Further analysis with gene specific primers indicated preponderance of cry1A, cry1B and cry1I genes and also of cry2Aa and cry2Ab. Results with few selected isolates (which were selected based on the bioassay results) indicated the presence of cry1Aa and cry1Ab genes. All these clearly indicated the specificity of these isolates for lepidopteran insect pests. The selection of local isolates will be useful in the generation of a transgenic plant with a local cry gene that offers resistance to the serious local pests. Hence PCR based techniques can effectively be used for characterization of B. thuringiensis strain collections.