Overexpression And Site-directed Mutagenesis Of Cry1Aa Gene From Bacillus Thuringiensis

Insects would develop resistance which correlated with the decreasing of pH of its midgut juice to Bacillus thuringiensis toxins, and the mechanism is unknown. A modified SDS-alkaline method was used to extract plasmids of Bt 4.0718 strain, and PCR analysis demonstrated that P3 plasmid contain crylAa gene. By analyze the consensus region of 13 kinds of cry\Aa gene with the National Center for Biotechnology information's BLAST WWW server, primers HOI and H02 were designed to amplified crylAa gene from P3 plasmid, then the PCR products subcloned to obtain recombinant MDAa strain. The 3.5-kb BamH I and Sal I insert of pMD18-T was inserted into the pET30a vector, then transformed E.coli strain BL21 (DE3) to giving ETAa strain, and a 141-kD fusion protein was overexpressed as inclusion body by induced with IPTGSite-directed mutagenesis was performed in a two-step PCR with three primers. The fragments produced in the first step were used as the "big primer" in the second PCR to amplified the 1048-bp fragments and subcloned. A 224-bp mutated fragment obtained by digesting pMDMX with Hhe I and BssH II and substituted the fragment in pMDAa at the same position to get recombinant plasmid pMDMa. pMDMa digested with BamH I and Sal I, the 3.5-kb mutated fragment was inserted into the pET30a vector to obtain the mutagenic strain ETMa C812A and C814A. SDS-PAGE showed that the mutant cryl Aa gene was also overexpressed.Inclusion bodies from ETAa strain and mutated ETMa strain dissolved under different pH or concentrations of 2-Mercaptoethanol individually. SDS-PAGE showed that, under the same pH or concentration of 2-Mercaptoethanol, inclusion bodies from mutated ETMa strain were more easy to dissolve, and could dissolve better in lowpH or low concentration of 2-Mercaptoethanol than inclusion bodies from ETAa strain. It demonstrated, that the mutagenesis of C812A and C814A could signally change the dissolvability of inclusion bodies. The study provide a valuable option for resistance management, and on such a base, the role of disulphide in the protoxin stability and toxicity can be further achieved.