[Effect of Cry1Ca7 protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hübner]

To obtain the mutants with different toxicity from the wild-type CrylCa7.

Insecticidal crystal protein CrylCa7 from Bacillus thuringiensis which is highly toxic to Spodoptera exigua, an important agricultural pest in China, and we mutated this toxin by over-lapping extensive PCR method in different domains to obtain 11 chimeric mutants.

The results of bioassays against Spodoptera exigua neonates showed that several conserved amino acid sites were crucial to insects. The pesticidal activities of most of mutated proteins were decreased, including Glycinel38 Serine, Threonine221 Aspartic acid, Threonine221 Argine, Asparagine251 Serine, 439GlycineGlycineThreonine440, Asparagine306 Argine, Tryp-tophan376 Phenylalanine, Argine522 Glutamic acid and Argine570 Glycine. The activity of those mutated proteins in the Domain II was 439GlycineGlycineThreonine440 < Asparagine306 Argine < Tryptophan376 Phenylalanine. In the Domain III, the mutant Argine522 Glutamic acid < Argine 570 Glycine, their toxicities reduced distinctly compared with CrylCa7. The toxicities of the mutant Argine148 Glycine in Domain I increased six-fold, nevertheless the activities of the mutants Glycine138 Serine, Threonine221 Argine and Asparagine251 Serine mutant reduced totally, even the mutant of Threonine221 Aspartic acid was not toxic entirely.

It is relatively easier to obtain mutant with higher toxicity in Domain I of CrylCa7 protein than these in both DomainII and III. We can use the improved mutant genes as the potential resources to construct novel engineering bacteria and transgenic plant, meanwhile, to perform the study of interaction mechanism between insects and Cry proteins.