Cry3Aa is a 73 kDa protoxin toxic to insect larvae of Coleoptera order. It is processed to form a stable 65 kDa & / #61540 / -endotoxin by endogenous proteases. The first part of this study involved the determination of the patterns of biosynthesis of Coleoptera-specific & / #61540 / -endotoxin by a local isolate of Bacillus thuringiensis subsp. tenebrionis (Btt) in relation to its growth and sporulation. Among four different media compared (DSM, GYS, HCT and C2) Cry3Aa production was the highest in DSM, especially at 72nd h and 120th h of incubation.
For improvement of Cry3Aa production, the effects of different carbon and nitrogen sources, inorganic phosphate and other mineral elements were tested. Increasing concentrations (5-10 g.L-1) of glucose or sucrose decreased the toxin yield probably by suppressing sporulation. Inorganic phosphate was found to have the most striking effect on toxin biosynthesis. 200 mM inorganic phosphate concentration resulted in 5 fold increase in Cry3Aa yield. Cry3Aa production was greatly reduced when various combinations of organic and inorganic nitrogen sources, especially ammonium sulphate and Casamino acids were replaced with Nutrient broth in DSM. The highest Cry3Aa production was obtained in the media containing 10-5-10-7 M MnCl2, 10-5 M FeSO4 and 5.10-4 M MgSO4, corresponding to their original concentrations in DSM. Decrease of iron concentration or its omission from the medium decreased the toxin yield.
Toxin production capacity of our local isolate was compared with those of 30 different anti-Coleopteran Bt strains. Most of the strains producing this protein gave general protein banding patterns quite similar to that of our local isolate.
Lastly, the cry3Aa gene of the Btt local isolate was PCR-amplified and cloned into the E. coli/Bacillus shuttle vector pNW33N. The recombinant plasmid was amplified in E. coli and the sequence of the cry3Aa was determined. Amino acid sequence deduced was found to be 97.4 %-99.2 % identical to the cry3Aa sequences (GenBank) of 10 different quaternary ranks. In this respect, the gene has to represent the 11th quaternary rank of the cry3Aa ones. The recombinant plasmid carrying cry3Aa gene was next used to transform Bs 168 as an intermediate host and low level of expression was seen.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12605910/index.pdf |
| Date | 01 February 2005 |
| Creators | Kurt, Aslihan |
| Contributors | Ozcengiz, Gulay |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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