Molecular Designing of Insecticidal Crystal Proteins Cry1Ca7 from Bacillus Thuringiensis
|School||Chinese Academy of Agricultural Sciences|
|Keywords||Bacillus thuringiensis Insecticidal Crystal Protein Site-directed Mutagenesis Over-lapping extensive PCR Cry1Ca7|
Bacillus thuringiensis is one of the most widely used insecticidal microorganism, Whose toxicity originate from the insecticidal crystal protein(ICP). The researches of the structure of ICP and the mechanism of toxin are the hotspots in the world. Baesed on the relation between protein structure and function, we can improve the toxin and change the specificity by the way of molecular designing, thereby providing new genetic resources for transgenetic plant and microorganism.The study includes a series of aspects: comparative modeling, molecular design, and the changes of receptor binding mechanism of mutants. The overall results are showed below:13 mutants by the over-lapping extensive PCR were obtained. They were expressed in the E. coli BL21 (DE3). R148G and F177S are located in the DomainⅠ. The bioassay showed R148G has an evidently increased toxicity,and F177S showed a slightly enhancement in toxicity. Others showed decreased toxicity or none. 439GGT440、N306R、W376F和P375F&P377Y are located in DomainⅡ. R522E和R570G are located in DomainⅢ. T221D、T221R、G138S and N251S are located in DomainⅠ. D12.3a and D12.3b/c lost the toxicity totally. D1123 and D1231 kept the activity.In the Bt expessive system, we obtained the BtF177S and BtF580S. The bioassay showed that the BtF177S has a slightly enhanced toxicity than the Cry1Ca7 and the BtF580S lost the toxicity totally. We purified the Bt Cry1Ca7、Bt Cry1Ca7+CT、Bt F177S and Bt F580S by HPLC.(1)The results of the competent experiment suggested that binding capability of mutants to BBMV became weaker than that of Cry1Ca7.(2)The oligomarization assay showed the mutants could bind to the BBMV and make the Cry toxin oligamarization.We transformed the cry1Fa2 into acrystalliferous strain, B. thuringiensis HD73 cry- by electroporation and made the bioassay to beet armyworm (Spodoptera exigua) and cotton bollworm (Helicoverpa armigera). The bioassay results showed that the toxicity to beet armyworm was the 8 percent of the Cry1Ca7, and the 10 percent of the Cry1Ac to H. armigera.It is significant of molecular design of ICP in theory and in practice. By the way of molecular design of Cry1Ca7, we obtained the mutants with improved toxicity which can be used for transgenetic plant and microorganism, and the mutants with decreased toxicity which can be used for the reseach of molecular mechanism.