Disulfide bond formation, reduction and isomerization are important posttranslational modification in proteins that occur in most, if not all, living organisms. In eukatoyes, disulfide bond in substrate proteins are primarily formes by ERO1 and PDI. ERO1, oxidized by molecular oxygen, acts as a specific oxidant of PDI, which then makes disulfide bonds in folding proteins oxidized directly. It means that ERO1 plays an essential role in setting the redox potential in the ER, and the regulation of Ero1p activity is critical to maintain redox homeostasis and proper ER folding activity. We have isolated and analysed a endoplasmic reticulum oxidoreductase (ERO1) from Bombye mori. It apperas that both an N-terminal CxxxxC motif and a C-terminl CxxCxxC motif are necessary for Ero1p fuction. In vivo, the result of the 5day of 5th instar larvae by RT-PCR and Real-Time PCR shows that posterior silkgland, skin and mid silkgland are revealed more than rhose of other tissues. The same result for tissue distribution of transcripts is appeared about ERO1 and PDI. In Bombyx mori, ERO1 is also supposed to correlate with PDI. Afterwards, more experiments are needed to figure out accurate interrelation between ERO1 and PDI.

Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

The nucleotide sequence of the Spodoptera litura granulovirus (SlGV) genome was determined and analysed. It was 124,121 bp long, with a 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 nucleotides or larger. The 133 putative ORFs covered 86.3% of the genome. Among these, 29 ORFs were conserved in most completely sequenced baculovirus genomes, 44 were granuloviruses (GVs)-specific, 4 were nucleopolyhedroviruses (NPVs)-specific, and 56 were present in some NPVs and/or GVs. Especially, we proved that there were 9 SlGV-specific ORFs in 44 GV-specific ORFs by RT-PCR. Chitinase and cathepsin genes involved in the liquefaction of the infected hostwere not found in the SlGV genome, which explains why SlGV-infected insects do not degrade in a typical manner. When the phylogenic relationship was analyzed using the nucleotide sequence of granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which were belonged to TypeI granulovirus.

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.

To develop an advanced baculovirus insecticide with additional advantages, such as higher toxicity and recovering to wild-type baculovirus, a novel recombinant baculovirus, NeuroBactrus was constructed. Bacillus thuringiensis crystal protein gene (cry1-5) and an insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of poyhedrin gene promoter, and by fusion of orf603 partial genes and AaIT under the control of early promoter of ORF3006 from Cotesia plutellae bracovirus. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by NeuroBactrus was occluded into the polyhedra, and activated as about 65 kDa of crystal protein when treated with trypsin. RT-PCR analysis indicated that transcription of AaIT gene occurs by 2 h postinfection (p.i.) and increased at 16 h p.i.. NeuroBactrus showed high toxicity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcNPV. Re-recombinants derived from NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro. This result showed that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 genes.

A new Bacillus subtilis isolate showed high anti-fungal activities (more than 80% control efficacy) against several plant diseases such as rice blast (Magnaporthe grisea), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans) and wheat leaf rust (Puccinia recondita). We tried to confer an insecticidal activity to this B. subtilis isolate for constructing a recombinant strain which has dual functions, anti-fungal and insecticidal activity. The insecticidal cry1Ac gene of B. thuringiensis was constructed under its own promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K-1Ac). The plasmid, pHT1K-1Ac was introduced into B. subtilis isolate by electroporation and the transformant was confirmed by PCR with cry1Ac specific primers. B. subtilis transformant produced a parasporal inclusion in the cells as in B. thuringiensis and the size of that protein was appox. 130 kDa. The insecticidal activity of the transformant was checked against lepidopteran pest, Plutella xylostella. This result suggests that this recombinant B. subtilis strain shows the possibility of controlling harmful insect pests as well as plant fungal diseases simultaneously at one crop, and both culture broth and harvested cells of this strain can be used as individual biological control agents separately for integrated crop protection.

As a continuation of a previous work by Park et al. (2006), we have developed a two-element radio interferometer that can measure both the phase and amplitude of a visibility function. Two small radio telescopes with diameters of 2.3 m are used as before, but this time an external reference oscillator is shared by the two telescopes so that the local oscillator frequencies are identical. We do not use a hardware correlator; instead we record signals from the two telescopes onto a PC and then perform software correlation. Complex visibilities are obtained toward the sun at λ=21cm for 24 baselines with the use of the earth rotation and positional changes of one element, where the maximum baseline length projected onto UV plane is ~90λ As expected, the visibility amplitude decreases with the baseline length, while the phase is almost constant. The image obtained by the Fourier transformation of the visibility function nicely delineates the sun, which is barely resolved due to the limited baseline length. The experiment demonstrates that this system can be used as a "toy" interferometer at least for the education of (under)graduate students.

Oral spirochetes are anaerobes known as one of causative agents for periodontal diseases. In this study, we investigated the possibility of utilizing fluorescent fatty acids for labeling oral spirochetes. Bacterial labeling was standardized with three different lengths of fluorescent fatty acids: 5-octadecanoylaminfluorescein (OAF), 5-dodecanoylamin-fluorescein (DAF), and 5-hexadecanoylaminfluorescein (HAF). Among these fatty acids, OAF showed the best labeling activity. Treponema denticola ATCC 35405 was totally saturated to the maximum when incubated with OAF 1μM/mℓ for 1 hour. Treponema vincentii LA-1 also increased in fluorescence in proportion to incubation time length and the concentration. In conclusion, these findings showed the possibility that the fluorescent fatty acid can be used for labeling oral spirochetes.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the geneitself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface asa GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by α3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing α3β4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

Bumblebees are important pollinators in greenhouse and have colonized all parts of the World. In Korea, the value of bumblebees is increasing as pollinator. However, the more recent use of reared colonies may ultimately allow pathogens to spread to peripheral areas for bumblebees. Generally, bumblebees are hosts to a large number of parasites which are mites, flies, protozoa, fungi, virus and bacteria. For most of these, very little is known about their effects against host insect, epidemiology or evolutionary ecology. So, we report pathogenic bacteria isolated from Bombus terrestris and B. ignitus at first time in Korea. Bacillus fusiformis and Klebsiella oxytoca are isolated from B. terrestris and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of B. fusiformis was 35~40% against B. terrestris. Pantoea dispersa and K. oxytoca are isolated from B. ignitus and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of these species were 35~40% against B. ignitus. These pathogenicity are considered as low-level.

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.

A strain of Bacillus thuringiensis, named Bt 1-3, was isolated from Korean soil sample and it showed high insecticidal activity against Plutella xylostella. Bt 1-3 was deterimined to belong to ssp. aizawai (H7) by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins. PCR analysis with specific cry gene primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2Ab genes. In addition, this isolate showed high uptake rate of foreign plasmid by electroporation. Based on these characteristics of Bt 1-3, we tried to construct a spore-free Bt 1-3 mutant by knock-out sigG gene, which is known as a key transcription factor during sporulation. First, we constructed a basal vector, named pDST, consisting of erythromycin resistant gene (EmR), partial polyhedrin gene and temperature sensitive origin of replication gene (Orits). Subsequently, according to the chromosomal DNA sequence of Bt subsp. konkukian 97-27, we amplifed upstream and downstream regions of Bt 1-3 sigG, and cloned into pDST (pDST-G). So far, several EmR colonies were obtained by electroporating into the wildtype Bt 1-3 and crossover by homologous recombination is going on.

The complete genomic nucleotide sequence of the Spodoptera litura multicapsid nucleopolyhedrovirus (SlMNPV) isolated in Korea, SlMNPV-K1, was determined. It was 137,435 bp long, with a 55.4 % A+T content and contained 132 putative open reading frames (ORFs) of 150 nucleotides or larger that showed minimal overlap. The 132 putative ORFs covered 87.7% of the genome. Among these, 131 ORFs were are homologous to genes identified in previously reported SlMNPV genome which consisted 139,342 bp and contained 141 putative ORFs. However, arrangement of some ORFs were somewhat different from each other. Even though the SlMNPV-K1 genome is smaller than that of previously reported SlMNPV genome and had lesser predicted ORFs, the main functional genes were all conserved. When the phylogenic relationship was analyzed using the nucleotide sequence of polyhedrin gene, SlMNPV-K1 was most closely related to Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) which were belonged to Group Ⅱ nucleopolyhedrovirus.

To develop an improved baculovirus insecticide with additional advantages, a novel recombinant baculovirus, AcB5B-AaIT was constructed. B. thuringiensis crystal protein gene (cry1-5) and insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin (polh) gene promoter, and AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus, respectively. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by AcB5B-AaIT was occluded into the polyhedra produced by the recombinant virus, and activated as about 65 kDa of crystal protein when treated with gut-juice of Bombyx mori. The AcB5B-AaIT showed about 50% reduced LT50 value compared to that of the recombinant virus, Ap1Ac, expressing Cry1Ac against Plutella xylostella larvae. In addition, Spodoptera exigua larvae fed the recombinant polyhedra of AcB5B-AaIT showed about 4 fold higher refusing diet effect compared S. exigua larvae fed the recombinant polyhedra of the recombinant virus, Ap1C, expressing Cry1C. AcB5B-AaIT could be transferred to wild-type baculovirus along with serial passage by the homologous recombination between two polyhedrin genes contained in polh-cry1-5-polh fusion protein gene. These results suggested that the novel recombinant baculovirus, AcB5B-AaIT, could be applied as advanced viral insecticide.

Among the cultured products of Beauveria bassiana SFB-205 (KCCM 10892P), the supernatant showed the highest insecticidal activity against 2nd instars of Aphis gossypii (Aphididae) nymphs under glasshouse condition. The enzymes in the supernatant were confirmed as active materials, and the chitinase was finally selected as a QC factor for commercial production. However, the chitinase activity in the supernatant decreased by 11-folds due to the thermal stress at 50℃ for 2 h. To obtain thermal stability, the chitinase in the supernatant was adsorbed to a precipitable material and the pellet was freeze-dried (PCT/KR2007/005886). The adsorbent-A showed about 92.7% of harvesting efficiency which was higher than the other candidates. The chitinase activity of the freeze-dried powder was kept up about 82.0% of initial activity for the same thermal stress. Subsequently, an optimal formulation recipe was established to maximize long-term storage stability and efficacy. SFB-205 oil-based formulation was stable up to 18 months at room temperature. It showed 96.1% efficacy against 2nd instars of A. gossypii nymphs at 1 day after the treatment in the glasshouse. This novel approach can be a promising method to develop competitive biopesticies in the entomopathogenic fungi, even though it needs to be intensively studied.