| Bacillus thuringiensis(Bt) has been used as a biopesticide in agriculture. The distinguished insecticidal characteristics of this bacterium are due to its ability for producing insecticidal crystal proteins(ICPs) during sporulation. Currently, it is important to study the molecular mechanism of insecticidal crystal in Bt for improving the yield of insecticidal crystal protein, improving the bioassay of Bt and constructing recombinant Bt strain with high efficiency and broad spectrum. However, the molecular structure and mechanism of Cry11 Ba needs further study.ClustalX2 and MEGA4.1 software was used to compare amino acid sequences of Cry11 Aa and Cry11 Ba, and DiANNA 1.1 Web Server was used to predict the position of Cry11 Ba disulfide bond, and Swiss-Model software was used to establish a 3D structural model of Cry11 Ba, of which five sites on cry11 Ba C-terminel and nine sites on N-terminel Domain I from 141 to 150 were selected to be mutated. Site-directed mutation on Cry11 Ba was carried out using the technology of oligonucleotide-induced circular mutation. The mutants were transduced and expressed in acrystalliferous Bt strain 4Q7. The biochemical characteristics of the mutated Cry11 Ba protein were studied through phase-contrast microscope, SDS-PAGE, solubility assay, trypsin stability analysis and bioassay.The five mutants in C-terminel could form oval crystals and express 81 kDa protoxins. Next, crystals protein of the five mutants increased gradually in buffer solution. Crystal protein of C714 S was more sensitive to trypsin. Furthermore, toxicities of these mutants against Culex quinquefasciatus obviously decreased compared to their parent protein. These results demonstrated that accumulated mutates of cysteine residues did not affect the formation of oval crystal and the efficient expression of protein. Furthermore, the solubility improved significantly may because of the introduced Ser residue with a hydrophilic group(-OH). According to the forecast results of disulfide bonds, Cys652 formate disulfide bond, and when disulfide bond destroy and spatial structure unstable, eventually leading to a significant bioassay decline in insecticidal crystal protein.The nine mutants in N-terminel could still form oval crystals. The size of crystals of I142 A decreased and G147 A, V148 A, I150 A could form one or two inclusion bodies, which may be caused by protein misfolding occurred in the formation process. I142 A expressed an approximately 60 kDa protein, probably because the mutation of this site could lead to the noncontinuity of gene expression, resulting in forming an uncomplete Cry11 Ba. The other mutants expresseed 81 kDa protoxins, indicating that these sites did not affect the expression. Except I142 A, crystal protein of the eight mutants has no significant difference in buffer solution. Trypsin stability analysis indicated that crystal protein of I142 A did not degrade into active fragments. However, the trypsin stability of crystal protein of Y145 A, E146 A, G147 A, S149 A was improved, indicating that this four sites may reduce sensitivity to trypsin. The bioassay results showed that G144 A were increased by 1.84 and 3.31 times to Culex quinquefasciatus and Aedes aegypti. The others were not toxic to these mosquitoes.In this research, 13 mutants were constructed with potential applied value, and discussion was deeply taken on the effect of cry11 Ba gene structure on insecticidal properties, providing evidence that the structure changes in crystal protein will affect its function. Simultaneously, the mutant G144 A with improving bioassay was obtained. This result provided technical support to construct Bt engineering strain with high efficiency, and also laid an important foundation in later selection and optimization of Bt mosquitocidal gene functional domains mutational sites, moreover, had great implications for development and application of microbial insecticides. |
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