Superexpression And Bioactivity Of Cry1Ac Gene From 20-kb DNA Associated With Cry1A Crystal Protein From Bacillus Thuringiensis

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but role and sequences of these DNA molecular is 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\ 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 crylAc gene 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 plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into Kcoli B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies by induced with IPTG 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 the 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 1.2X2.0 um Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against third-instar larvae ofPlutella xylostella, the lethal concentrations (after 48 h) of larval exposure of the former was 20.5 fig/ml, and which of the latter was 6.25 fig/ml.. DNA fragments extracted from inclusion bodies of ETX53 strain and crystals of HTX42 strain were used as template individually, PCR analysis demonstrated that these 20-kb DNA fragments had polymorphism.All these made a good ground for constructing insecticidal recombinants and analyzing the source, structure, and function of the 20-kb DNA.