ABSTRACT
In order to increase the production of insecticidal crystal proteins Cry1 and Cry2, firstly, Plack-ett-Burman design was applied to evaluate the effectiveness of the related nutrition factors; it was found that the soybean powder and MnSO4?H2O were significant factors for Cry1 production, but the yield of Cry2 wasn’t effected remarkably in such medium. Then the steepest ascent experiment was adopted to approach the optimal region of the medium composition. Lastly, the optimal concentration of the soybean powder and MnSO4?H2O was 11.5 and 0.02 g/L, obtained by response surface methodology (RSM). The final yields of Cry1 and Cry2 was 0.32 mg/mL and 0.11 mg/mL, increasing twice more than that in the medium optimized before. The median lethal concentration (LC50) of optimal medium was 1.09 ?L/mL. The toxicity to Heli-coverpa armigera was significantly enhanced than the old one.
ABSTRACT
The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.