Isolation of a Bacillus thuringiensis strain from South African soils and the characterization of its cry gene sequence.

[摘要] The objectives of this project was to isolate and characterize a Bacillus thuringiensisstrain from South African soils, determine its cry gene sequence, clone this genesequence and determine its toxicity. Forty four putative Bacillus thuringiensis strainswere extracted from soil samples taken from the Muldersdrift mountain range, theRoodekrans botanical gardens, Southbroom in Kwazulu Natal and Nelspruit in easternMpumalanga province. The bacterial populations of these soil samples were isolated andclassified using different microbial, and biochemical techniques including sodium acetatetests to isolate putative B. thuringiensis spores. These spores were cultured and furthercharacterized through colony shape and colour as well as the presence of δ-endotoxincrystals. Once characterized, DNA was extracted from the isolates using an array oftechniques to obtain high quality template DNA. This DNA was then screened via PCRusing truncated versions of the cry1A specific primers TYIAA (f) and TYIUN12 (r). Theinsecticidal protein CRY1A was selected for this study since it is specific and highlytoxic to lepidopteron insects. Homology to the cry1a gene was detected in six of theBacillus strains analyzed, namely S4, S9, S10 n1, n3 and n5. PCR products were clonedinto the pTZ57R/T cloning vector and transformed into JM109 competent cells. DNAfrom 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 (approximatelyl,500-bp) fragments from many bacterial genera confirmed that the isolates were indeedBacillus thuringiensis, showing evidence of lineage according to the signature sequenceswithin the conserved regions. Spore/δ-endotoxin mixtures of the randomly selectedisolate S10 were used in a bioassay to test their toxicity against the lepidopteron insectGalleria 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.

[发布机构] University of the Witwatersrand