Journal of Applied Biosciences (J. Appl. Biosci.) [ISSN 1997 - 5902]
Volume 31: 1878 - 1886. Published July 8, 2010.
Molecular characterization of Bacillus thuringiensis strains with differential toxicity to the spotted stalk borer, Chilo partellus
1Opondo S. O., 1Ngure, R. M., 2Nguu, E. K. and 2Ochanda J.O.and 2Ogoyi, D.O*.
1Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536, Njoro. Kenya. 2Department of Biochemistry, University of Nairobi, P.O. BOX 30197, Nairobi, Kenya.
*Corresponding author email: dogoyi@uonbi.ac.ke
ABSTRACT
Objective: Three Bacillus thuringiensis isolates designated 1M, K10-2 and V24-M with varying levels of toxicity to Chilo partellus (spotted stalk borer) were characterized to establish the basis for the differential toxicity and to identify any unique properties that may be used to screen other isolates.
Methodology and results: Bt isolate colonies were picked from plates and inoculated into LB medium and growth rate estimated by measurement of cell density at absorbance 600nm. Secreted proteins was quantified and analysed through SDS-PAGE. Bt plasmid DNA was isolated and restricted with different restriction enzymes and analysed by electrophoresis. Isolate 1M had a slightly elevated growth rate in LB medium at the lag and exponential phases of growth than both isolates K10-2 and V24-M , but at the plateau phase the growth rates were not significantly different for all the three isolates. All crystals were bi-pyramidal in shape suggesting that the Bt isolates are leptidopteran specific. The major protein bands for isolate K10-2 and V24-M had molecular weights of 28, 65, and 130kDa while main protein bands in isolate 1M were of molecular weight 28 and 65kDa. The three Bt isolates showed varying levels of plasmid DNA concentration with isolate 1M having the highest plasmid concentration suggesting greater plasmid copy number. Upon restriction digestion of the isolatesplasmid DNA with EcoRI, BamHI and HindIII, one particular unique band of 7200 bp was evident in all the digests.
Conclusion and application of findings: Bt isolate, 1M which had the highest toxicity against C.partellus in a previous study showed a unique protein profile and higher plasmid copy number than the other isolates. The results suggest that these properties may be used for selective identification of Bt isolates that exhibit effective toxicity against C. partellus.
Key words: Bacillus thuringiensis, δ- endotoxin , plasmid, differential toxicity, Chilo partellus.
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