The objectives of this project was to isolate and characterize a Bacillus thuringiensis
strain from South African soils, determine its cry gene sequence, clone this gene
sequence and determine its toxicity. Forty four putative Bacillus thuringiensis strains
were extracted from soil samples taken from the Muldersdrift mountain range, the
Roodekrans botanical gardens, Southbroom in Kwazulu Natal and Nelspruit in eastern
Mpumalanga province. The bacterial populations of these soil samples were isolated and
classified using different microbial, and biochemical techniques including sodium acetate
tests to isolate putative B. thuringiensis spores. These spores were cultured and further
characterized through colony shape and colour as well as the presence of δ-endotoxin
crystals. Once characterized, DNA was extracted from the isolates using an array of
techniques to obtain high quality template DNA. This DNA was then screened via PCR
using truncated versions of the cry1A specific primers TYIAA (f) and TYIUN12 (r). The
insecticidal protein CRY1A was selected for this study since it is specific and highly
toxic to lepidopteron insects. Homology to the cry1a gene was detected in six of the
Bacillus strains analyzed, namely S4, S9, S10 n1, n3 and n5. PCR products were cloned
into the pTZ57R/T cloning vector and transformed into JM109 competent cells. DNA
from the six isolates was also characterized at the 16S rDNA level with the use of PCR.
Primers capable of amplifying nearly full-length 16S ribosomal DNA (approximately
l,500-bp) fragments from many bacterial genera confirmed that the isolates were indeed
Bacillus thuringiensis, showing evidence of lineage according to the signature sequences
within the conserved regions. Spore/δ-endotoxin mixtures of the randomly selected
isolate S10 were used in a bioassay to test their toxicity against the lepidopteron insect
Galleria mellonella. No significant mortalities were reported, but sensitivity to the S10 δ-
endotoxin was evident when compared to results using known B. thuringiensis δ-
endotoxins at the same concentrations.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/3920 |
| Date | 23 October 2007 |
| Creators | Laridon, Neil Edward |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2237061 bytes, application/pdf, application/pdf |
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