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.

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.

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.

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. -casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of -casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. -casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of -casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine -casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.