누에씨는 매년 계대사육을 통해 자원을 보존하지만, 이 과정에서 잠종의 소실 및 혼합사고 등의 위험이 있어, 누에 유전자원의 효율적이고 안전한 장기보존법 개발이 필요한 상황이다. 본 연구에서는 2년 동안 저온에 보관 된 누에 보급품종(백옥잠, 대황잠, 백황잠)과 누에 유전자원(n29, sa2, yang2)의 누에씨를 봄과 가을 사육기에 맞추 어 점진법을 사용하여 부화를 유도하였다. 부화된 누에씨의 부화비율과 함께 전령 경과기간, 화용비율, 전견중 등의 사육 성적을 조사하였다. 2년간 저온 보관된 누에씨의 부화비율은 보급품종에서 87~88%, 유전자원에서는 71~75%로 나타났다. 화용비율은 보급품종에서 79~89%, 유전자원에서는 71~79%로 조사되었다. 품종 지정 시 사육 성적과 비교해 볼 때, 부화비율, 화용비율, 번데기 무게, 고치무게 모두 감소하는 경향을 보였다. 또한, 2년 동안 저온에 보관된 후의 누에씨 부화기간은 1년 동안 저온에 보관된 누에씨보다 1~2일 더 길었다.

To verify the progenitor of B. mori, we sequenced 14 B. mori strains preserved in Korea and one B. mandarina collected in Korea and conducted phylogenetic analysis of Bombycidae using maximum-likelihood method and concatenated sequences of 13 PCGs and 2 rRNA genes. All B. mori strains, regardless of their origin, formed a strong monophyletic group, with the highest nodal support. This B. mori group was placed as the sister to the two B. mandarina collected each from Korea and Shandong, China with the highest nodal support. Finally, the remaining two B. mandarina, which were collected in Japan were independently placed as the most basal lineage of B. mori and B. mandarina group. These results appear to indicate that an immediate ancestor for the domestic silkworm strains may have been originated from China and Korea.

Bombyx mandarina (Lepidoptera: Bombycidae) is generally regarded as the ancestor of the domesticated B. mori. Recently, over 40 mitochondrial genomes (mitogenomes) mainly from B. mori strains preserved in China and wild individuals of B. mandarina were sequenced to verify the progenitor of B. mori. At this point, we also were curious about the origin and the relationships of Korean silkworms to foreign B. mandarina and B. mori. As a first step, we sequenced the complete mitogenome of the B. mandarina collected in Korea and compared it to pre-exsiting data (37 strains of B. mori and 14 individuals of B. mandarina). The complete mitogenome of B. mandarina was 15,694-bp long, consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one non-coding region. The 494-bp long A+T-rich region possessed the highest A/T content (95.3%) than any other region of the genome. Overall, the general mitogenome characteristics of the genus Bombyx species have an identical gene arrangement, similar A/ T content (average 82.3%) and so on. Phylogenetic analysis, however, showed that B. mori and B. mandarina formed a distant group each with the highest nodal support. For more findings of mitogenome characteristics of Bombyx including the Korean B. mandarina and those preserved in Korea more mitogenomes, particularly from Korea, might be needed.

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.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

Our previous study demonstrated that Coprisin, a peptide from Copris tripartitus infected with bacterial pathogens, has an antibacterial activity. We assessed in this study whether Coprisin caused cellular toxicity in various mammalian cell lines. Coprisin selectively caused a marked drop of cell viability in Jurkat T cells, U937 cells and AML-2 cells belonging to the human leukemia cells but not in Caki cells and Hela cells. Fragmentation of DNA, a maker of apoptosis, was also confirmed in theleukemia cell lines but not in other cells. The Coprisin-induced apoptosis in leukemia cells was mediated by AIF (apoptosis inducing factor), a caspase -independent pathway.

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.

COPRISIN is an antibiotic substance extracted from Copris tripartitus. This study is intended to identify various cell biological stimuli that COPRISIN, widely known as an antibacterial substance, has on human cells and to identify its molecule mechanism. A variety of human cell lines were divided into epithelial cells including kidney cells or womb cells, and immunocyte including T cells or macrophages and, after their being cultivated and maintained, cell biological changes of the respective cells according to COPRISIN treatment were compared. As a result, it was confirmed that, different from other experiment cells, COPRISIN specifically caused cell kill in T cells and macrophages. That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to COPRISIN treatment. An Apoptosis process is one dependent upon activity of caspase family protein, it was proved that COPRISIN medium cell kill process was one through a caspase-independent route such as AIF. Though it was found out that transcription of TNF-α and extracellular TNF-α secretion increased in blood cells stimulated by COPRISIN, it was also confirmed that TNF-α was a major medium factor in a COPRISIN induced cell kill process from the fact that a cell kill process by COPRISIN was not inhibited at all with TNF-α inhibiting antibody treatment. Above results revealed that COPRISIN, different from other tissue origin cells including kidney cells, can specifically induce apoptosis in immunocyte, which is caused by a caspase-independent cell signal transmission route.

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.