The Influence Of Site-direct Mutation On The Toxicity Of Bacillus Thringiensis Cry1Ba3 Against Plutella Xylostella

Bacillus thuringiensis (Bt for short) is a kind of microbial insecticide most widely studied and used so far. The insecticidal activities of Bt come from the parasporal crystal proteins produced when Bt grows up, which is called insecticidal crystal proteins also (ICPs for short). It has been a central issue to research the relationship between the amino acids, the structures of ICPs and its activities. Cry1Ba3 is the insecticidal crystal protein with high toxicity against Plutella xylostella revealed by our laboratory. In order to find amino acids significant for its activity, this study analyzed Cry1Ba3 with steps as below.A homology model was conducted in the database Swiss—Model to build a model of 3D-structure of Cry1Ba3, and its three main domains were figured out Preliminary. The first amino acid to the 270th is DomainⅠ; DomainⅡlocates between 280 and 480 while DomainⅢstays in 500~630. Three hydrophobic regions which contain 60~80, 260~263, 397~404 were found out after analyzing Cry1Ba3 with software BioEdit. At last, 40 sites were selected to mutate after multiple sequence alignment between Cry1Ba3, Cry1Aa1, Cry2Aa1, Cry3Aa1 and Cry4Ba1, 40 mutants were expressed in Escherichia coli (E. coli for short) BL21 (DE3) after being constructed by semi-overlapping primer PCR. The bioassay results of Plutella xylostella indicates that the toxicity of mutant M6 (R171L),M10 (W282A),M19 (H485G) decreased significantly. The activity of mutant M6 (R171L) has losed completely and the activity of mutant M10 (W282A) and M19 (H485G) decreased 49 times and 44 times respectively.The secondary structures of all mutated protein were predicted after submitting their sequences to the website P?le Bio-Informatique Lyonnais. The results suggested that the structures of mutants whose activities unchanged changed barely.This research seeked out three key amino acids those are important to the insecticidal activities of Cry1Ba3. This achievement will provide reference on methods and theories for the mechanism researches. What's more, it may help to synthesize new type broad-spectrum insecticidal crystal proteins with high toxicity. At last, this achievement will increase productability of agriculture and save the souce used on research.