| Cry3A protein of Bacillus thuringiensis spores secreted by δ-endotoxins, can kill the Coleoptera insect specifically. In this study through expression of Cry3 A containing α-chymotrypsin digestion sites, insecticidal activity core fragment aCry3 A was obtained. Existing of polymer ion channel was verified in the lethal process of Cry3 A toxin. Effect of α1-α3 in non-core area of Cry3 A toxin is explored and methods of increasing Cry3 A insecticidal activity are acquired.Specific methods are as follows: pET28a-cry3 am plasmids conserved in lab were transferred into competent cells for expressing mutant protein Cry3Am3; Toxic core fragment of 55 kD was obtained after digestion with α-chymotrypsin. of Cry3 Am According to MgCl2 extraction method of settlement of lepidoptera brush border membrane vesicles(BBMV), BBMV of Tenebrio molitor was separated successfully. Hypothesis is demonstrated by Western Blot that Cry3 A can form polymer protein channel in BBMV. Larvae of Tenebrio molitor was fed with Cry3 Am, Cry3 A and feed and lethality of mutant protein Cry3 Am is evaluated. Effect of α1-α3 fragment in noncore area of Cry3 A toxin is explored.The result was that active fragment aCry3 A forms shadows in the 110 kD, 165 kD and 225 kD region in the photograph of Western Blot after chemiluminescent.In compound feed in mixed-case of 0.12% toxin, lethality rate of Cry3 Am is 22.6% while it was 24.1% of aCry3 A. Lethality rate of Cry3 A was 3.7%. Based on study of the combination characteristics of cry3 A and midgut BBMV of Tenebrio molitor, Cry3 A toxin was found to form a polyprotein channel in the lethal process which will destroy the osmotic equilibrium of insect midgut cells. α1-α3 fragment in non-core areas of Cry toxin had no effect on the formation of polyprotein channel; mutant protein Cry3 Am with α-chymotrypsin digestion sites can be inserted between aCry3 A and α1-α3 of non-core region,can improve the insecticidal activity.. |
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