To examine the expression profile of oxidative stress responsive genes in Spodoptera litura, we constructed a cDNA library from S. litura injected with hydrogen peroxide (H2O2). Using a microarray chip composed of 2,964 cDNAs, we screened gene expression at 1, 3, 5, 7, and 9 h post H2O2 injection. Data were clustered into 15 groups of genes that behave similarly across each time course. Seventy-three genes were identified as being at least 2-fold up- or downregulated after treatment with H2O2 in S. litura. We constructed expressed sequence tags (ESTs) for genes that changed at least 2-fold after treatment with H2O2. The functional classification of these ESTs based on Gene Ontology showed that the ESTs are rich in genes involved in oxidoreductase activity (5.7%), defense (14.3%), cellular process (22.9%), and development (17.1%).

For stable germline transformation, the promoter of B. mori cytoplasmic actin gene (BmA3) was used to ubiquitous expression of transgenes. Except for BmA3 promoter, promoters used to regulate gene expressionin all tissues and developmental stages of B. mori were not nearly developed. To identify more powerful promoter than previously reported BmA3 promoter (Mange et al., 1997), we introduced a new dot blot hybridization method, and isolated nine clones that show stronger dot signal compared to the control, BmA3by this method. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This resulting positive clone, named bHsp70 (B. mori heat shock protein 70) was ubiquitiously expressed in tissues and developmental stage of fifth instar B. mori larvae,and stimulated bythermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5'-flanking region of bHsp70 gene that has 264-fold more intensive promoter activity than BmA3 promoter. Moreover, transcription activity of bHsp70 promoter under heat shock condition (42 ℃, 4 hr) was increased over 2-fold than normal condition. Therefore, we suggest that bHsp70 promoter may be used more effective candidate for transgene expression in B. mori.

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.

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the slow cis/trans isomeraization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. We studied the characterization of Cyclophilin A (bCyp A) isolated from Bombyx mori . The cDNA of bCyp A is 947 bp. There is a 5´-untranslated region of 91 nucleotides followed by an initiating ATG codon. The TAA termination codon occurs at nucleotide 588. Thus translation of the sequence from nucleotides 91 to 588 would produce a protein of 166 amino acids with a calculated molecular mass of 18.2kDa. The 'AATAAA' consensus polyadenylation signal and poly A tail are present in the 3´-untranslated region. To analysis of PPIase activity, we expressed the bCyp A protein in Sf9 cell by using baculovirus expression vector system (BEVS). SDS-PAGE and Western blot analysis showed that the molecular weights of intracelluar expressed protein was approximately 28.2 kDa. The PPIase activity assay was monitored by proteolytic cleavage of the chromophore p-nitroanilide byα-chymotrypsin. As substrate the synthetic tetrapeptide succinyl-Ala-Ala- Pro-Phe-p-nitroanilide was used.

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant nuecin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.

정확한 in vitro 전사가 일어날 수 있는 진딧물의 세포추출액을 제조하였다. 전사를 직접 조절할 수 있는 단백질 인자를 규명하기 위하여 전사개시점과 그의 상류에 결합하는 DNA 결합단백질을 탐색했다. 전사개시점을 포함하는 단편 A(-194/23)에는 52kDa, 50kDa, 40kDa의 단백질들이 결합했으며 전사개시점 상류의 DNA 단편 B(-393/-263)에는 52kDa, 50kDa, 40kDa의 단백질들이 결합한 반면 DNA 단편 C(-263/-195)는 53kDa단백질만이 결합했다. 그리고 이들 DNA 결합단백질들의 DNA 결합 활성에는 양이온이 요구되었다.