Isolation of a Bacillus thuringiensis strain from South African soils and the characterization of its cry gene sequence.
Year: 2007
Type: dissertation
Abstract: The objectives of this project was to isolate and characterize a Bacillus thuringiensis \nstrain from South African soils, determine its cry gene sequence, clone this gene \nsequence and determine its toxicity. Forty four putative Bacillus thuringiensis strains \nwere extracted from soil samples taken from the Muldersdrift mountain range, the \nRoodekrans botanical gardens, Southbroom in Kwazulu Natal and Nelspruit in eastern \nMpumalanga province. The bacterial populations of these soil samples were isolated and \nclassified using different microbial, and biochemical techniques including sodium acetate \ntests to isolate putative B. thuringiensis spores. These spores were cultured and further \ncharacterized through colony shape and colour as well as the presence of δ-endotoxin \ncrystals. Once characterized, DNA was extracted from the isolates using an array of \ntechniques to obtain high quality template DNA. This DNA was then screened via PCR \nusing truncated versions of the cry1A specific primers TYIAA (f) and TYIUN12 (r). The \ninsecticidal protein CRY1A was selected for this study since it is specific and highly \ntoxic to lepidopteron insects. Homology to the cry1a gene was detected in six of the \nBacillus strains analyzed, namely S4, S9, S10 n1, n3 and n5. PCR products were cloned \ninto the pTZ57R/T cloning vector and transformed into JM109 competent cells. DNA \nfrom the six isolates was also characterized at the 16S rDNA level with the use of PCR. \nPrimers capable of amplifying nearly full-length 16S ribosomal DNA (approximately \nl,500-bp) fragments from many bacterial genera confirmed that the isolates were indeed \nBacillus thuringiensis, showing evidence of lineage according to the signature sequences \nwithin the conserved regions. Spore/δ-endotoxin mixtures of the randomly selected \nisolate S10 were used in a bioassay to test their toxicity against the lepidopteron insect \nGalleria mellonella. No significant mortalities were reported, but sensitivity to the S10 δ- \nendotoxin was evident when compared to results using known B. thuringiensis δ- \nendotoxins at the same concentrations.
Author: Neil Edward Laridon
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W2249006053