| Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemical pesticides. Genetic improvement of Bt strains, offers a promising means of providing technology and strain resources to develop novel Bt-based bioinsecticide products.In order to obtain Bt strains with enhanced toxicity and large-spectrum to improve the application of Bt insecticides, we designed and synthesized the DNA sequence of insect-specific VGCCs blockerω-ACTX-Hvla according to the codon preference of the Bt strain, and fused it to the3'-end of insecticidal crystal protein gene cry1Ac. Then we investigated the expression, crystallization, and functional activity of the fusion protein in Bt strain. The fusion gene of cry1Ac,ω-ACTX-Hv1a and enhanced green fluorescent protein gene egfp was expressed in Bt acrystalliferous strain Cry-B under the control of the native gene crylAc expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell. A166kDa full-length fusion protein was identified by immunoblot analysis. This convinced that the fusion gene could be intactly expressed in Bt strain. The bioassayes of lysis of recombinant strain indicated that, virulence of the fusion inclusions was at least fivefold higher than CrylAc crystals toward larvae of Spodoptera exigua. It was also toxic to Helicoverpa armigera larvae and significantly retarded the growth of the larvae. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in Bt by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity. The study provides an important foundation for constructing fusion protein with different insecticide activity and recombinant strains with higher toxicity.Although the expression of cryl genes has been widely studied, the mechanisms of crystal formation and assembly in Bt strains remain unclear, because it is relatively difficult to study the assembly and aggregation of Cry proteins in individual cells in vivo. In this work, we used the engineering strain Cry-B(1Ac-ACTX-EGFP) as the object, observed crystallization of the fusion protein in samples collected at consequent time point with confocal laser scanning microscopy. The result showed that, at the beginning of expression, the fusion protein was distributed in the mother cell. The formation of crystal is a process that can be compared to protein molecule polymerization. Surprisingly, the fusion protein crystals showed polarity and were located near two ends of the strain. Before cell lysis, the crystals grew to full size. This reporter system may be a useful tool to study the mechanisms of parasporal crystal formation and assembly.Then, a comparative proteomic analysis was performed on the Bt engineering strain Cry-B (1Ac-ACTX-EGFP) and Cry-B (1Ac) before cell lysis using label-free quantitative shotgun proteomics to investigate the underlying regulators participating in formation of insecticidal protein crystals. In total,201proteins from Cry-B (1Ac-ACTX-EGFP) and138proteins from Cry-B (1Ac) were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, in which,114proteins were shared by the two strains. The result of protein functional categorization demonstrated that, the proportion (in percentage) of identified proteins related to oxidation-reduction showed great changes between the two strains. This indicated that the different oxidation-reduction environment inside the cell may be one of reasons caused crystal difference between the two strains. We found the proteins with notable difference in expression level between the two strains and the proteins with a high emPAI value only identified in one strain, such as60kDa chaperonin (GroEL) and10kDa chaperonin (GroES) involved in preventing protein's misfolding and promotes the refolding and assembly; Clp protease involved in cell's response to stress; catalase that catalyzing reduction of hydrogen peroxide and so on. These proteins may be the regulators participating in crystals' formation. Certainly we still need do more molecular experiments and combine the GFP reporter system to test and verify the relationship between the function of these proteins and the mechanisms of crystal formation. This work provided valuable technologic information and data for exploring the mechanisms of crystal formation and further improving Bt strains by rational application of genetic engineering.
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