Heterogenous Expression And Genetical Modification Of Insecticidal Crystal Proteins From Bacillus Thuringiensis

Bacillus thuringiensis, a widely distributed Gram-positive, spore-forming,aerobic bacterium, has been the most used biological pesticide due to its specificactivity, which is mainly contributed from the crystalline inclusion body formedduring sporulation composed of one or more insecticidal crystal proteins (ICPs, orδ-endotoxins), against target insects with environmental safety.In this paper, the heterogenous expression of ICPs from B. thuringiensis as wellas the regulation analysis in an aquatic recipient strain and the genetical modificationof ICPs were studied to improve the activity and persistence of the toxins againstlepidopteran and dipteran larvae.The cyt1Aa gene from Bti, whose product synergizes other mosquitocidal toxinsand functions as repressor of resistance developed by mosquitoes against Bacilliinsecticides, solely and alongside the cry11Aa gene from Bti as well, were introducedinto the aquatic Gram-negative bacterium Asticcacaulis excentricus, living in upperwaters as a natural food resource for mosquito larvae, who has been proved as asuccessful host for Bacilli mosquitocidal toxins. The genes were introduced as anoperon but, although DNA sequencing verified correct and mRNA was detected forboth genes, no Cyt1Aa toxin was detected even though cyt1Aa solely was expressedwell in another operon. Both Cyt1Aa and Cry11Aa were expressed using a constructin which a promoter was inserted upstream of each gene. The necessity of twopromoters for co-expression of cyt1Aa and cry11Aa in A. excentricus could becorrelated with the special RNA and restriction-modification system of A. excentricus.Recombinant A. excentricus expressing both toxins was found with nearly the samepace in toxicity to 3rd instar larvae of Culex quinquefasciatus but with slower growingpace, ultimately to be 2.21- and 12.6-fold as toxic as the recombinant expressing just Cry11Aa against susceptible and resistant C. quinquefasciatus larvae, respectively,suggesting that the expressed Cyt1Aa burdens the growth of, but synergizes Cry11Aaand thus increases the toxicity of recombinant A. excentricus cells. Furthermore, thisrecombinant A. excentricus co-expressing cry11Aa and cyt1Aa was less sensitive toultra-violet radiation than wild Bti stain was, potentially acting as a candidate forfurther mosquito control.In addition, hybrid toxins Cry1CAA and Cry11ABB, which were constructed byreplacing domainsⅡ-Ⅲof Cry1Ca and Cry11Aa by the corresponding domains ofCry1Ab and Cry11Bb, respectively, were expressed in Bt acrystalliferous strain.SDS-PAGE and Western blot showed that the recombinant strains could express twohybrid proteins in recombinant strains during sporulation, with molecule weight andimmunogenicity identical to the original wide-type toxins. Importantly, the twoproteins has improved activity to the tested insects than their origins. ProteinCry1CAA was 0.558- and 3.26-fold toxic to neonate Helicoverpa armigera larvaethan Cry1Ab and Cry1Ca respectively, and 4.08- and 0.355-fold toxic to neonateSpodoptera exigua than the two origins, respectively. The toxicity of hybrid toxinCry11ABB was not significantly higher than wide-type proteins, with LC50 values of339.2 and 245.1 ng/ml to susceptible and resistant third-instar larvae of C.quinquefasciatus, respectively, corresponding to 40 % and 25 % increase of toxicitythan Cry11Aa. It implies that domain swapping technology might be applied for thegenetical modification ofB. thuringiensis toxins.