A large number of transgenic crop varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins have been commercialized in 13 countries since 1996. Although the use of these insect-resistant Bt crops can increase crop quality and yields, concerns remain about the potential negative effects of such crops on ecosystems. Transgenic soybean containing cry1Ac gene have been developed to control Lepidopteran pests of soybean and we aimed to investigate whether this soybean could affect non-target arthropods, which play a major role in ecological functions in agricultural ecosystems. In the present study, we first measured the levels of Cry1Ac protein in Bt soybean at different growth stages of soybean and then we compared the community structure of arthropods occurred in fields of transgenic and wild-type soybean. The levels of Cry1Ac protein in transgenic soybean leaves ranged from 252.9 to 604.5 μg g-1 DW. Multivariate analyses (PerMANOVA and NMDS) showed that the composition of the non-target arthropod community was affected by sampling date but not by soybean genotype. These results suggest that transgenic soybean expressing Cry1Ac protein may not adversely affect such non-target arthropod communities.

The cry gene from Bacillus thuringiensis (Bt), encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

Under field conditions, we investigated how transgenic Bt cabbage expressing the insecticidal Cry1Ac1 protein affects two target Lepidoptera species — Plutella xylostella (Plutellidae) and Pieris rapae (Pieridae) — as well as the structure of the local non-target arthropod community. When exposed to Bt cabbage line C30, Plutella xylostella and Pieris rapae were significantly less abundant than when in the presence of the non-transgenic control. Multivariate analyses (PerMANOVA and NMDS) showed that composition of the non-target arthropod community was affected by sampling date but not by cabbage genotype. These results suggest that transgenic cabbage expressing Cry1Ac1 protein can be effective in controlling Plutella xylostella and Pieris rapae in the field and that cultivation of this cabbage may not adversely affect such non-target arthropod communities.

Six of cadherins have been selected from the P. xylostella genome 52 open reading frames are annotated as cadherin-like genes. Compared to other 5 cadherins of P. xylostella (PxCads), PxCad1 has the highest homology with other lepidopteran insect cadherins and PxCad1 was expressed in all developmental stages specially in gut tissue. Expression of PxCad1 was suppressed by feeding its specific double-stranded RNA (dsRNA, 150ng/larva) and treatment of dsPxCad1 significantly reduced susceptibility to Bt Cry1Ac toxin. To confirm the specific interaction between PxCad1 and Cry1Ac, a toxin-binding assay was performed using enzyme-linked immunosorbent assay (ELISA). The ELISA indicates that BBMV extracted from PxCad1-silenced P. xylostella have significantly lower binding activity to active form of Cry1Ac than control BBMV. Moreover, the analysis of the binding parameters showed that the toxin affinity (Kd) of the control BBMV extract (BBMV-dsCON) was 6.08 ± 0.84 nM, which was not much different to the affinity value (6.72 ± 0.81 nM) of the dsPxCad1 treatment. However, there was a remarkable difference in number of binding sites (Bmax), in which BBMV-dsCON extract had 1.61 ± 0.04, but the BBMV-dsPxCad1 extract had 0.88 ± 0.02. Taken together, these results are suggest that PxCad1 is a functional receptor for Cry1Ac toxicity against P. xylostella larva.

Transgenic lines of insect resistant cabbage (Brassica oleracea var. capitata) expressing Cry1Ac1 protein has been developed to control diamondback moth (Plutella xylostella). The potential adverse effects of Bt crops on non-target arthropod herbivores and predators are major concerns. We conducted a tritrophic bioassay to study the ecological impacts of insecticidal transgenic cabbage on the wolf spider (Pardosa astrigera), a non-target generalist predator. First, we measured the levels of Cry1Ac1 proteins in fruit flies that were fed with the transgenic cabbage as well as those levels in the wolf spiders preying on the Bt cabbage-fed fruit flies using enzyme linked immunosorbent assay (ELISA). Cry1Ac1 proteins were detected in the Bt cabbage fed fruit flies and also in the wolf spiders after preying on Bt cabbage-fed fruit flies. Second, we compared the life history characteristics of the wolf spiders preying on the Bt or non-Bt cabbage. Growth, development time and survival of the wolf spiders were not significantly different between Bt and non-Bt cabbage. Although the wolf spiders were exposed to Cry1Ac1 protein via feeding on the preys containing Cry1Ac1 proteins, their growth and survival was not significantly affected.

Putative cadherin genes, which are a receptor of the Bacillus thuringinesis toxins, were predicted from a whole genome sequencing data from the diamondback moth, Plutella xylostella. After the sequence and expression analysis, a Bt receptor cadherin gene was selected. The P. xylostella cadherin gene (PxCad1, GenBank Accession no. GU901158.1) encodes 11 cadherin repeats and a transmembrane domain. The PxCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of PxCad1 gene was suppressed by feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly influence on pupal and adult development of P. xylostella. However, the larval treated with dsRNA PeCad1 (150 ng/larva) significantly reduced susceptibility to B. thuringiensis Cry1Ac (4.83 μg/ml). By contrast, the dsRNA PxCad1 -treated larvae did not show any change in susceptibility to B. thuringiensis Cry1Ca (0.24 μg/ml). These results suggest that PxCad1 is a specific receptor of Cry1Ac toxin from B. thuringiensis in P. xylostella.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic crops resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (95.6%) to those of Cry1Ac which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues on domain I and II. In order to convert these residues to Cry1-5 randomly, 10 mutagenic primers were designed. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBI-Modcry1Ac based on cry1-5 and constructed 63 mutant cry genes. Among them, 10 mutant cry genes on domain II were selected and their recombinant proteins were expressed by baculovirus expression system. From bioassay results to P. xylostella and S. exigua, we found some mutants have high insecticidal activities to be applicable to transgenic crops.

The effects of pollens of Bt Chinese cabbage (Brassica campestris), expressing the insecticidal protein Cry1Ac toxin (Bt Chinese cabbage) on Bombix mori larvae, were determined in laboratory studies. When reared on an artificial diet containing pollens of Bt Chinese cabbage, decreased survival rate and body weight of B. mori larvae were observed. ELISA test using Cry1Ac-antibody implied that the Cry toxin was detected not only in the whole body and alimentary canal but also in the hemolymph and remaining body parts, suggesting that the ingested Cry1Ac protein is distributed inside the body through epithelial membrane of the midgut of B. mori. Taken together, it was suggested that the pollens of Bt Chinese cabbage adversely affect non-target insect B. mori larvae.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic plants resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (97.9%) to those of Cry1Ab which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues in domain Ⅰ and domain Ⅱ, and we focused on domain Ⅰand domain Ⅱ regions and designed 10 mutagenic primers to change 12 residues. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBⅠ-Mod-cry1Ac based on cry1-5 and constructed 63 various mutant cry genes. In the further study, we will express those mutant proteins as a fusion form with polyhedrin using baculovirus expression system and subsequently do bioassay to Spodoptera larvae.

Bacillus thuringiensis(Bt) crystal protein (Cry1Ac) genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis(Bt) crystal protein genes. We transformed three difference Cry1Ac (Cry1Ac and two modified Cry1Ac) genes into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR, and RT-PCR. We generated 30 independent lines of transgenic soybean plants. Analysis of the flanking sequences isolated by Inverse PCR revealed complex T-DNA insertion patterns and preferential integration of T-DNA into the intergenic spacer region of the soybean genome. We found 5 different intergenic transgenic soybean lines of soybean genome. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis, physiology test, and agronomic characters are investigating.

Cry1Ac protein is known as one of toxin crystal proteins synthesized from Bacillus thuringenesis that plays a critical role for the insect resistance. Recently, cry1Ac genes have introduced into many plants in general and soybean as well. However, the gene expression of cry1Ac genes in transgenic plants remains low that need to be improved. Several mutations we reintroduced into the cry1Ac genes in order to enhance the insecticidal effect. In this study, the cry1Ac with mutant #2, #11 and #16 were transformed into Kwangan, a Korean soybean variety by using the “half-seed” method. The plant lets carrying modified cry1Ac genes were primarily selected on media containing Phosphinothricine (PPT), a bar selective agent and Basta leaf painting. Then, the presence of introduced genes in T0 plants and the gene expression were investigated by PCR, RT-PCR and Real-time PCR. PCR and RT-PCR analysis showed expression of bar and cry1Ac genes from tested transgenic soybean plants. The number of copy of bar gene ranged from 1 to 3 by Real-time PCR analysis. These results provided a fundamental back ground for our further experiments: Confirmation of the gene expression by Southern blot and identification of the function of modified cry1Ac by insect bioessays.