Influence of cry2A sporulation-dependent promoter and molecular chaperone ORF1-ORF2 from Bacillus thuringiensis on Cry11Aa protein

Abstract:

[Objective] To analyze the coordination function of cry2A sporulation-dependent promoter and the enhanced expression of molecular chaperone ORF1-ORF2 to crystal protein Cry11Aa. [Methods] We constructed three recombinant plasmids pHcy1, pHcy2 and pHcy4 containing cry11Aa gene. pHcy1 carried cry11Aa own promoter and p19 gene, and pHcy2 carried cry2A sporulation-dependent promoter and orf1-orf2 gene. pHcy4 inserted cry2A promoter and orf1-orf2 gene upstream pHcy1 plasmid. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of Bacillus thuringiensis sub sp. israelensis. We performed SDS-PAGE to analyze Cry11Aa protein expression in the recombinant Bt strains and carried out the mosquitocidal bioassay. [Results] SDS-PAGE showed that Cry11Aa protein was detected in 4Q7(pHcy1) and 4Q7(pHcy4), but not in 4Q7(pHcy2). The cry11Aa gene could not be regulated under cry2A promoter. Cry11Aa protein had a 1.25 fold expression amount in the equal volume culture of 4Q7(pHcy4) to that of 4Q7(pHcy1), which indicated that molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent. Both 4Q7(pHcy1) and 4Q7(pHcy4) formed Cry11Aa crystals in a similar size and shape during sporulation under the transmission electron microscope. Their LC50s against 3rd-instar Culex quinquefasciatus were 59.33 ng/mL and 66.21 ng/mL respectively. [Conclusion] Whether crystal protein from B. thuringiensis could successfully express might relate to the type of the used promoter and their coordination. Molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent with an unknown mechanism, but did not have an effect on high mosquitocidal toxicity of Cry11Aa protein. This research might play an important role to search the best collocation between ICP promoter or chaperone gene and ICP gene and to construct high-toxic Bacillus thuringiensis engineering strain by chaperone gene.

Key words:Bacillus thuringiensis; molecular chaperone; p19 gene, orf1-orf2 gene; crystal protein Cry11Aa