The differences in adult lifetime among various silkworm strains has been suggested that the adult lifetime may be genetically controlled. In this experiment, using J037 and Daizo strains we investigated genetic factors related to the adult lifetime of silkworm. We constructed the full-length cDNA library from the adult male of the J037 strain. A total of 2,688 clones were randomly selected, and we performed a differential display hybridization with cDNA probes generated from J037 and Daizo adult males. In conclusion, 193 clones were identified as differential expressed genes, and 154 unique genes were generated after the assembly of 193 clones. Of the 154 unique genes, the most abundant genes were cytochrome oxidase subunit-1 gene(9 times) and unknown(clone ID; 1-50) gene(5 times). The functional groups of these unique genes with matches in the AmiGo database were constructed according to their putative molecular functions. Among thirteen functional categories, the largest group was unclassified protein(24%). In addition, We analyzed the nucleotide and deduced amino acid sequences of the most highly occurred gene(1-50, EF434397), which consisted of 240 amino acids. However, it is confirmed yet that these genes really have an affected on the silkworms longevity.

The antimicrobial peptide cecropin was isolated from the larval hemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. The full-length cDNA of A. yamamai cecropin (Ay-cecA) was cloned by a combination of RT-PCR and 3' RACE based on N-terminal sequence obtained by Edman degradation. The cloned cDNA consists of 419 nucleotides encoding a 64 amino acid precursor containing a 37-residue mature peptide. Like many insect cecropins, Ay-cecA also harbored a glycine residue for C-terminal amidation at the C-end. To understand this peptide better, we successfully expressed bioactive recombinant Ay-cecA in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The fusion CecA-KSI protein was expressed as inclusion body at high level. Recombinant Ay-cecA was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr), and purified by FPLC chromatography. The purified recombinant Ay-cecA showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. The time-kill assay showed that Ay-CecA displayed a time-dependent bactericidal activity, as was also seen after treatment with melittin. our results proved that Ay-cecA can be developed into novel antibacterial agent.

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.

Inflammatory bowel disease (IBD) is a group of chronic disorders of unknown etiology characterized by inflammation of the gastrointestinal tract. Recent data showed that the development of IBD is associated with the interplay of genetic, bacterial, and environmental factors and dysregulation of the intestinal immune system. We investigated how the gut cells were repaired after injury in Drosophila melanogaster. In this study we made D. melanogaster intestine damage model by oral feeding with variety IBD inducer such as pathogenic bacteria Serratia marcescens, Dextran Sulfate Sodium (DSS) and bleomycin, because its function is very similar with human, even though D. melanogaster has relatively simple organism. We repeated oral feeding with variety IBD inducer and got the survival rate and 50% lethal dose (LD50). After feeding with IBD inducer, we investigated the change of the intestinal stem cells, innate immune-related gene expression, and apoptosis in D. melanogaster gut. We examined the Delta, stem cell marker, staining image in the gut after feeding with DSS and S. marcescens with LD50 concentration. The Delta positive cells greatly increased in gut cells damaged by DSS or S. marcescens. This result supports the idea that intestinal gut stem cells are increased after gut cell damage and play very important role in damaged cell repair. Expression level of antimicrobial peptides was dramatically up-regulation after gut damage. As a result of TUNEL (terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling) assay, we confirmed that cell death by apoptosis was very increased in DSS feeding flies. Accordingly, we suggest that D. melanogaster is a proper IBD model organism to study how intestine damage can be repaired.

For the purposes of this paper, stress may be defined as any modification of environmental parameters that leads to a response by biological organisms. Stresses that affect biological structures may be non-thermal, such as ultraviolet radiation, pH, or salinity, or thermal. Temperature is one of the major stresses that all living organism face. The major effects of cold shock are decrease of membrane fluidity and the stabilization of secondary structures of RNA and DNA in the cells, which may effect the efficiency of translation, transcription, and DNA replication. In this study in compliance with the cold temperature stress about selection of the useful gene is contents from the silkworm which is been revealed. The survival rate which is caused by with the cold temperature stress until 12 hours was 100% in 0℃, until 2 hours was 100% in -5℃. A total of 960 clones were randomly selected from the subtraction cDNA library, and then performed a differential display hybridization analysis with forward and reverse probes. In conclusion, selected 53 partial clones and novel 2 full-length clones were identified as differential expressed genes. We assumed that the novel gene related with transmembrane.

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.

Electroporation is well known today as a powerful transfection technique and is useful for the study of gene expression. The advantage of the electroporation method is that large quantity of silkworm (Bombyx mori) eggs can be transformed in a very short time. However, how to use it for introducing foreign gene into silkworm eggs needs systematical investigation. In our silkworm transgenesis program, we needed an efficient technique to evaluate the functionality of transgenes before their injection into eggs. The goal of this experiment was to find an alternative efficient method of generating transgenic silkworm eggs using a commercially available electroporation device. The Gene Pulser Xcell (Bio-Rad Laboratories, USA) were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology. We investigated pigmentation-rate and hatching-rate of the silkworm eggs of electroporation. We used foreign gene LacZ, EGFP, Ds-red induced vector system with selection marker for transgenic silkworm. The LacZ gene in 3rd instar larva DNA can be detected by β-galactosidase stain. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is available technique for transfecting B. mori egg.

SAGE technique is a sequenced-based approach that identifies which genes are expressed and quantifies their level of expression. The SAGE catalog of gene expression for a given cell or tissue is defined as the 'transcriptome'. With a goal of obtaining a set of quantitative information of expressed genes of posterior silk gland (PSG) of silkworm, we have generated a SAGE tag library from the PSGat day 4 of 5th instars of Bombyx mori. In this study, atotal of 2,406 tags were identified, representing 682 unique transcripts. Of these SAGE tags, 1,982 tags were detected twice or more accounted for 82% of the total tag population, whereas 445 tags were detected only once accounted for 18% of the total tag population. Four percent (27 tags) of the unique tags were detected at least ten times each, which corresponds to a representation of more than 53% of the total tag population. In addition, we have discussed a comparative aspect of the transcript abundance between expressed sequenced tags (ESTs) and the SAGE tags.

본 연구는 동충하초속균의 자실체 인공 재배를 위한 기초자료를 얻고자 제주도 한라산에서 채집한 Cordyceps longissima, C. militaris, C. pruinosa 등 야생 동충하초의 형태적, 생리적 특성조사를 수행하였다. Cordyceps longissima : 지좌는 숙주에서 1~2개 발생하며 곤봉형 또는 불규칙형으로 표면이 다소 거칠고 적갈색을 띤다. 두부는 23~59×6~8 ㎜이며, 병부는 원통형으로 37~151×2.5~4.0 ㎜이다. 자낭각은 묻힌형으로 553~600×215~270 ㎛. 자낭은 가는 원통형으로 330~510×5~6 ㎛이며, 자낭포자는 11~13×1.4~1.8 ㎛이다. 자실체는 매미목 매미과의 곤충에서 발생하였다. 균사 생장은 MCM 배지에서 25℃, 4주 배양시 73 ㎜이며, 균사의 색택은 진황색을 나타내었다. 탄소원으로는 단당류인 glucose, 질소원으로는 potassium nitrate를 가장 잘 이용하였다. C. militaris : 지좌는 숙주에서 1~3개 발생하며 곤봉형으로 주황색을 띤다. 두부는 9~23×6~8㎜이며, 병부는 원통형으로 27~30×6~7 ㎜이다. 자낭각은 반묻힌형으로 465~510×260~310 ㎛. 자낭은 가는 원통형으로 320~385×4.2~4.8 ㎛이며, 자낭포자는 2.2~4.5×1.2~1.4 ㎛이다. 자실체는 나비목 곤충의 번데기에서 발생하였다. 균사 생장은 PDA 배지에서 25℃, 4주 배양시 71 ㎜이며, 균사의 색택은 연노랑색을 나타내었다. 탄소원으로는 단당류인 frucose, 질소원으로는 potassium nitrate를 가장 잘 이용하였다. C. pruinosa : 지좌는 숙주에서 1개 발생하며 곤봉형으로 주홍색을 띤다. 두부는 7~9×3~4이며, 병부는 원통형으로 13~22× 2~3 ㎜이다. 자낭각은 반묻힌형으로 350~520×130~310 ㎛. 자낭은 원통형으로 256~270×5~6㎛이며, 자낭포자는 3.2~5.1×0.8~1.3 ㎛이다. 자실체는 나비목 곤충의 번데기에서 발생하였다. 균사 생장은 PDA 배지에서 25℃, 4주 배양시 60 ㎜이며, 균사의 색택은 연붉은색을 나타내었다. 탄소원으로는 단당류인 arabinose와 mannose, 질소원으로는 sodium nitrite를 가장 잘 이용하였다.