Molecular characters and induction profiles of HIF-1α and other bHLH-PAS domain-containing proteins identified in a carcinogenic liver fluke Clonorchis sinensis

오규석 가천대학교 대학원 2019 국내석사

Clonorchis sinensis, a trematode parasite that invades the hepatobiliary tracts of vertebrate hosts with limited available oxygen requires a significant amount of oxygen for its sexual reproduction and energy metabolism. However, little is known about molecular mechanisms involved in the maintenance of oxygen homeostasis and/or adaptation to the hypoxic environments in the liver fluke. In this study, we characterized molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix-loop-helix and Per-Arnt-Sim (bHLH-PAS) domain-containing proteins such as HIF-1β, single-minded protein (SIM), and aryl hydrocarbon receptor (AHR), which may prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed functional domains that are tightly conserved in each of the bHLH-PAS subfamilies, whilst the oxygen-dependent degradation domains could not be defined in the probably partial C. sinensis HIF-1α (CsHIF-1α) protein. Expression of the CsHIF-1α gene was highly induced in worms either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite also affected the CsHIF-1α expression depending on the surrounding oxygen tension. Treatment of a prolyl hydroxylase inhibitor under normoxia condition resulted in an increase of the CsHIF-1α level. Conversely, the other bHLH-PAS genes were less responsive to these exogenous stimuli. Taken together, our data suggest that the HIF-PHD-VHL triad constitutes a key regulator of physiological responses to hypoxia also in trematodes including C. sinensis. To our best knowledge, this is the first report concerning identification and characterization of HIF-1α and relevant proteins in the phylum Platyhelminthes. Clonorchis sinensis는 제한된 이용 가능한 산소로 척추 동물 숙주의 간 담관을 침범하는 흡충류 기생충으로 유성생식과 에너지 대사에 상당한 양의 산소가 필요하다. 그러나 산소 항상성의 유지 또는 담관 내에서와 같은 저산소 환경에서의 적응에 관련된 분자 기전에 대해서는 알려진 바가 거의 없다. 본 연구에서는, hypoxia-inducible factor-1α (HIF-1α)와 basic helix-loop-helix 및 Per-Arnt-Sim (bHLH-PAS) 도메인을 포함하는 단백질인 HIF-1β, single-minded protein(SIM) 및 aryl hydrocarbon receptor (AHR) 등의 분자 구조와 유도 특성을 확인하였고, 이들은 C.sinensis에서 저산소증에 대한 적응을 돕는 것으로 확인되었다. 이들 단백질은 bHLH-PAS 서브 패밀리 각각에서 강하게 보존되는 기능성 도메인을 보유하고 있는 반면, C.sinensis HIF-1α (CsHIF-1α) 단백질에서는 산소 의존성 분해 도메인을 부분적으로 규명 할 수 없었다. CsHIF-1α 유전자의 발현은 저산소 상태에 노출되거나 인간 담관 세포와 함께 배양 된 간흡충에서 높은 정도로 유도되었다. 산소 이외에 산화 질소와 아질산염 또한 주변 산소 분압에 따라 CsHIF-1α 발현에 영향을 주었다. 정상 산소 상태에서 수산화억제제(prolyl-4-hydroxylase inhibitor)를 처리한 경우에는 CsHIF-1α 발현이 증가하였다. 반면에 다른 bHLH-PAS 유전자들의 경우, 이러한 외부 요인들에 대해 비교적 덜 반응하였다. 결론적으로, 본 연구에서는 C. sinensis를 포함한 흡충류에서도 HIF-PHD-VHL triad가 저산소증에 대한 생리적 반응의 중요한 조절자라는 사실을 확인하였다. 이 연구는 편형동물에서 HIF-1α 및 관련 단백질의 동정 및 특성을 규명한 최초의 연구이다.

Analysis of proteome in clonorchis sinensis adult worms and identification of antigenic protein by immuno-proteomics

이명노 Graduate School, Korea University 2013 국내박사

Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. I identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, I found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. These proteins will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies.

Blanching에 의한 햇순나물의 이화학적 특성 및 기능성 변화

김민하 영남대학교 대학원 2012 국내석사

Clonorchis sinensis infestation induces 3-dimensional invasion of cholangiocarcinoma

Jihee Won 고려대학교 대학원 2014 국내석사

Clonorchis sinensis (C. sienensis) is a worm parasite that is prevalent in East Asian countries, and it causes chlonorchiasis, diverse symptoms occurred by infection of C. sinensis. Since epidemiological correlation between C. siensnsis infestation and CCA was reported, initiation of CCA involved in C. sinensis has been researched in many experimental research cases such as current studies suggesting that excretory-secretory products (ESPs), molecules that are secreted by this parasite and released into surround hepatobiliary tissue, play important role in host-parasite interaction related to CCA initiation, whereas progress of CCA has not been. Now, I investigated the possibility that C. sinensis infestation affects on poor prognosis of CCA by inducing the invasion of CCA. In the CCA cells with ESPs treatment, expression of focal adhesion proteins and secretion of matrix metalloproteinases (MMPs), related to mobility of the cells, were increased. Furthermore, Using an advanced CCA culture models based on a novel microfluidic assay, I quantitatively addressed the role of ESPs gradients from C. sinensis in promoting the invasion of CCA cells into neighboring ECM.

Establishment, expression and characterization of Miscanthus sinensis transformed by Agrobacterium tumefaciens using COMT gene related to lignin biosynthesis : 리그닌 생합성 관련 유전자 COMT 발현특성 구명 및 Agrobacterium을 이용한 참억새 형질전환체계 확립

JiHyeYoo 강원대학교 일반대학원 2016 국내박사

Miscanthus sinensis is a diploid hybrid and a temperate, perennial, cross-pollinating grass used as bioenergy plant, biomass production and high quality cellulose and ethanol production. The establishment of EST data base is important to study classification and function of gene groups in Miscanthus sinensis. A cDNA library from Miscanthus after UV-B treatment was prepared from mature mRNAs, and 1,000 cDNA clones were partially sequenced. A total of 827 unigenes, including 72 contigs and 755 singletons, were identified after assembly of the expressed sequence tags (ESTs). To analyze the ESTs from the UV-induced cDNA library, grouped the sequences and classifications based on the best homology match of BLASTX searches against various plant protein sequences. The functional classification sequences belonged to 15 categories including metabolism, energy, cell cycle, transcription, protein synthesis, protein fate, protein with binding function, regulation of metabolism and protein function, cell transport, signal transduction, cell defense, interaction with the environment, cell fate, biogenesis of cellular components, and subcellular localization, which were considered to be of interest for UV-response signaling. Firstly, I isolated the caffeic acid O-methyl-transferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, I produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidase expression, which are related to the phenyl-propanoid pathway and antioxidant activity. The MsCOMT down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT down-regulated transgenic plants. I also observed a morphological difference with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway. This study was carried out to enhance the transformation rate from Miscanthus sinensis, to characterize the agronomic and morphological traits, the expression of MsCOMT in transgenic plant, the variation in lignocellulosic component, and the variation in phenolic compounds content. An efficient transformation of callus from M. sinensis was established using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish stable transformation system, I obtained high transformation rate from callus by various transformation factor explant type, strain, co-culture periods, acetosyringone concentration, and selective marker. Finally, in this study, seven putative transgenic plants were obtained. Through various tests including PCR analysis and southern blot were to detect antisense of COMT digested Xba I and Sac I restriction enzymes. The biomass of the control plant was superior than transgenic plants. Total lignin content of transgenic plants was lower than the control plant, due to the reducing of COMT gene expression related to lignin production. Cellulose and hemicellulose contents in transgenic plants were not increased. Variations in cellulose and hemicellulose content had no correlation with variations in lignin content of transgenic plants. Total phenolic compounds content was generally lower in the stem of transgenic plants than in the leaf. In the control plant, the ratio of phenolic compounds content in leaves and stems was almost similar. 참억새(Miscanthus sinensis)는 국내에 흔히 자생하는 다년생 C4 식물이며, 비농경지 지역에 서식하는 영년생 잡초로 알려져 있으나, 건물수량이 ha당 20 ~ 40톤 정도로 매우 높아 바이오에탄올 생산을 위한 원료작물로 활용하고자 하는 연구가 활발히 이루어지고 있다. 그러나 참억새의 높은 리그닌 함량은 바이오에탄올로의 전환효율을 감소시켜 실용화에 장벽이 되고 있다. 따라서 본 연구는 리그닌 생합성 관련 유전자인 COMT (Caffeic acid O-methyl-transferase)의 발현특성을 구명하고 Agrobacterium을 이용하여 참억새의 형질전환체계를 확립하고, 형질전환체의 생육특성 및 성분분석, 리그닌 함량의 변화를 확인하였다. UV를 처리한 참억새로부터 mRNA를 분리하여 cDNA library를 제작하였으며, EST 분석 결과 metabolism 관련 유전자 35개 에너지 관련 유전자 24개 세포주기 및 DNA 과정 관련 유전자 4개, transcripton 관련 유전자 14개, 단백질 합성 관련 유전자 21개, Protein fate 관련 유전자 54개, 공통인자 및 연결 기능 단백질 관련 유전자 223개, 단백질 기능 조절 관련 유전자 29개, 세포상호작용 시그널 관련 유전자 7개, 식물방어작용 관련 유전자 24개 등으로 유전자들의 기능을 분류하였다. 이중 리그닌 및 에너지 관련 대사 유전자들을 유용유전자로 선별하였고 MsCOMT로 명명하였다. 참억새 내에 있는 COMT 유전자의 full length 염기서열을 얻었고 NCBI blast를 이용하여 타작물의 COMT와 유사성을 분석하였다. COMT를 down regulation 시켜 유전자의 기능을 억제하여 벡터를 제작하였고, 클로닝된 MsCOMT 유전자를 이용하여 모델식물인 담배와 참억새에 형질전환을 시도하였으며, 그 체계를 확립하여 COMT 유전자의 발현 및 기능을 구명하였다. 형질전환 체계 확립은 식물체부위, strain, 공동배양 기간, acetosyringone 농도, 선발마커 등 다양한 요인을 적용하여 연구하였다. 미숙화기와 종자에 Agrobacterium strain LBA4404를 접종 한 후 3~4일간 공동배양할 시 캘러스 형성율 및 재분화율이 증가하였으며, kanamycine 선발마커와 aceto-syringone 200 uM에서 재분화율이 증가하였고, 참억새 형질전환체는 7개체를 얻었다. 형질전환체 참억새의 초장 및 줄기 직경, 엽장, 엽폭, 잎 세포 크기 등이 대조구에 비해서 작은 형태적 특징을 나타냈다. 형태적으로 왜소한 형질전환체는 페놀 및 플라보노이드 함량도 대조구에 비해 낮았으며, 이는 모델식물인 담배에서도 비슷한 결과를 나타냈다. 모델식물 담배 형질전환체에서 MsCOMT-AS 유전자가 발현할수록 리그닌 생합성에 관여하는 유전자인 APX, PAL의 발현량이 줄어드는 것을 확인하였다. MsCOMT-AS 유전자 기능으로 인하여 참억새 형질전환체에서 리그닌의 함량이 감소하는 것을 확인하였으며, 줄기와 잎 부분에서 대조구에 비해 약 50% 정도 감소되는 것을 확인할 수 있었다. 반면에 셀룰로스 및 헤미셀룰로스의 함량은 대조구와 차이가 없는 것을 확인하였다. 연구결과 COMT 유전자는 리그닌 합성에만 직접적인 영향이 있는 것으로 사료되며, 셀룰로스와 헤미셀룰로스 합성에는 관여 정도가 적은 것으로 사료된다.

Purification and preliminary crystallization of 14-3-3 protein from clonorchis sinensis

조용욱 Graduate School, Korea University 2013 국내석사

Clonorchis sinensis 14-3-3 proteins (C.sin14) are considered the antigenic proteins of C. sinensis which is widespread in Asian countries, including the Repbulic of Korea and China. The consumption of raw freshwater fishes is one of the major factors for C.sinensis infection. Since the infection of C. sinensis can affect people’s health in several ways, diagnostic methods for the C. sinensis infection are needed to prevent serious health problems, such as the development of cholangiocarcinoma. In this study, due to the possibility that the C.sin14 can be tagetted for a vaccine, we attempted to determine the C.sin14 structure. The recombinant 14-3-3 protein from C. sinensis was overexpressed, purified and crystallized. The C.sin14 purified by protein purification methods including chromatographic techniques was used for protein crystallization. For the primary step to determine the structure of C.sin14, I performed the crystallization by using hang-drop vapour diffusion method. I had grown the appropriate crystals at two optimal conditions. By using precipitant 1.10 M Ammonium tartrate (pH 7.0), the appropriate crystals were formed and the optimal concentration of the C.sin14 was about 20 mg/ml. The crystal grew to size about 0.2 mm x 0.2 mm x 0.1 mm within one week. The second condition is 0.14 M sodium fluoride, as a precipitant 20 % (w/v) polyethyleneglycol 3,350 (pH 7.3). The size is about 0.1 mm x 0.2 mm x 0.1 mm within two weeks.. A diffraction data set was collected to 3.0 Å resolution by using synchrotron radiation at photon factory BL1A beam line in Japan. The crystals of C.sin14 belong to the space group P432, P4132, P4332 or P4232 and a solvent content is 54.6%. The crystals belong to the cubic space group, with unit-cell parameters a=b=c=221.922 Å, and α=β=γ=90°. The asymmetric unit contains 6 molecules, corresponding to a Matthews coefficient (VM) Value of 2.71 Å3 Da-1. Determination of the protein structure is currently in progress.

Anti-inflammatory effects of ethanolic extracts of Chaenomeles sinensis leaves : 모과잎 에탄올 추출물의 항염증 효과

한영기 건국대학교 일반대학원 2015 국내석사

The Chaenomeles sinensis fruit is commonly used to treat conditions such as pneumonia and bronchitis, but a dearth of research on the leaf exists. In this study, we investigated the anti-inflammatory effects of C. sinensis leaf extract (CSL) on LPS-stimulated RAW 264.7 cells. The 70 % ethanol extract of CSL (CSLE) significantly inhibited NO, IL-6, and TNF-α production; additionally, the CSLE suppressed LPS-stimulated inducible NOS (iNOS), IL-1β, and phospho-STAT1 expression. In this study, we investigated whether CSLE decreases the production of inflammatory mediators through the inhibition of the TRIF-dependent pathways. Our results suggest that CSLE attenuates LPS-stimulated inflammatory responses in macrophages via the regulation of the TLR4 pathways, providing scientific support for its traditional use in the treatment of a variety of inflammatory diseases. Chaenomeles sinensis는 일반적으로 폐렴, 기관지염 등에 사용된다. 본 연구에서는 lipopolysaccharide로 자극시킨 RAW 264.7세포에서 Chaenomeles sinensis 잎 추출물의 항 염증 효과를 조사하였다. 그 결과 Chaenomeles sinensis 잎 70% 에탄올 추출물이 NO 및 iNOS 생성을 억제 시켰다. 또한 Chaenomeles sinensis 잎 70% 에탄올 추출물은 IFN-β 및 인산화된 STAT1의 생성을 억제 시켰다. 위 결과를 바탕으로 TRIF-dependent 경로의 염증성 매개체 생성 억제 확인실험을 하였다. 실험 결과 Chaenomeles sinensis 잎 70% 에탄올 추출물은 LPS로 자극되어 생성된 염증성 사이토카인인 TNF-α, IL-6 및 IL-1β를 억제 시켰다. 연구 결과를 종합하면 Chaenomeles sinensis 잎 70% 에탄올 추출은 LPS로 자극된 반응물들을 감소시키며, 염증 메커니즘을 조절 할 수 있는 가능성을 과학적인 증거로 제시한다. 또한 이 연구를 바탕으로 염증성 질병에 치료제로써 사용할 가치가 있다고 보여진다.

간흡충 항원 유전자의 클로닝 및 재조합 항원을 이용한 혈청학적 진단

심서보 연세대학교 대학원 2002 국내석사

토끼의 담도로부터 얻은 살아 있는 간흡충을 protease inhibitor cocktail이 들어 있는 RPMI배지에 넣고 유지하여 분비배설항원을 얻었다. 이 분비배설항원 중 21 kDa 부위의 단백질을 면역시킨 흰쥐의 혈청과 간흡충증환자 혈청으로 간흡충 성충의 cDNA library를 immunoscreening하여 강하게 반응하는 양성 clone을 선별해 내었다. 간흡충증 환자 혈청을 이용해 분리한 2개의 clone중 하나는 9개의 amino acids가 23회 반복되는 구조를 포함하는 265개의 amino acids로 구성되었고 glycine의 비중이 높아 GRCSP2라 명명하였다(GenBank Accession number AF461709). 다른 하나는 10개의 amino acids가 22회 반복되는 구조를 포함하는 269개의 amino acids로 구성되었고 proline의 비중이 높아 PRCSP라 명명하였다(GenBank Accession number AF461711). 간흡충 분비배설항원을 면역시킨 흰쥐의 혈청을 이용해 분리한 4개의 clone은 모두 흡충류의 myoglobin과 유사성이 높아 C. sinensis myoglobin이라 하였고 이는 150개의 amino acids로 구성 되었다(GenBank Accession number AF461710). GRCSP2와 PRCSP는 간흡충 total RNA 상에서 모두 1.05 kb 크기로 발현되었고 C. sinensis myoglobin은 0.57 kb 크기로 발현되었다. Southern blotting 결과 3가지의 유전자 모두 간흡충 genomic DNA에 존재함을 확인하였다. GRCSP2와 PRCSP의 recombinant protein을 purification하여 간흡충증의 진단에의 민감도를 Western blotting으로 검사해 본 결과 각각 93.3%와 86.7%를 나타냈으며 폐흡충증, 요코가와흡충증, 회충증, 스파르가눔증 환자 혈청에 교차반응을 보였고, 특히 PRCSP가 더 많은 교차반응을 나타냄을 확인하였다. Clonorchis sinensis adult worms obtained from the biliary tract of an experimentally infected rabbit were suspended in RPMI including a protease inhibitor cocktail in order to obtain C. sinensis excretory-secretory(ES) antigens. A C. sinensis cDNA library was screened with immune rat sera which were immunized with the 21 kDa protein of ES antigens and also screened with C. sinensis-infected human sera. Two clones were isolated by using C. sinensis-infected human sera. The cDNA insert of one clone contained a single open reading frame(ORF) of 795 base pairs encoding for 265 amino acids including 23 tandem repeats of 9 amino acids. Because glycine was a major component in its content, the protein was named GRCSP2(GenBank Accession number AF 461709). The cDNA insert of the other contained a single ORF of 807 base pairs encoding for 269 amino acids including 22 tandem repeats of 10 amino acids. Because proline was a major component in its content, the protein was named PRCSP(GenBank Accession number AF 461711). Four clones were isolated by using the immune rat sera against 21 kDa protein of ES antigens. The cDNA inserts of four clones were very similar and contained a single ORF of 450 base pairs encoding 150 amino acids. Using BLAST program, we could find significant similarity between the cloned gene and a trematode myoglobin gene such as Paramphistomum epiclitum, so the protein was named C. sinensis myoglobin(GenBank Accession number AF 461710). Northern blotting was performed to examine the expression of the cloned gene in C. sinensis. When total RNA from C. sinensis was hybridized to the GRCSP2 gene, PRCSP gene, and C. sinensis myoglobin gene, a single band of 1.05 kb, 1.05 kb, and 0.57 kb was detected respectively. The results of Southern blot hybridization indicated that each cloned gene was present within the C. sinensis genome. Purified recombinant GRCSP2 and PRCSP showed high sensitivity for serodiagnosis, 93.3% and 86.7%, respectively. But the cross-reactivity of each recombinant protein was found against paragonimiasis, metagonimiasis, ascariasis, and sparganosis patients' sera. The rate of cross-reactivity using purified recombinant PRCSP was more higher than that using purified recombinant GRSCP2.

Egg‐specific expression of DNMT2 with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis

HYE-JEONG CHO 가천대학교 대학원 2016 국내석사

Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both ofwhichwere elevated in the C. sinensis eggs.However,CsDNMT2and 5 mCweremarginally detected in other histological regions ofC. sinensis adults including ovaries andseminal receptacle. Themethylationsite seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon.Taken together, our data strongly suggest that C. sinensis has preserved the functional DNAmethylation machinery and thatDNMT2 acts as a genuine alternative toDNMT1/DNMT3 to methylateDNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

곤충유전자원 분석을 위한 데이터베이스 구축 및 활용에 관한 연구

조용훈 전남대학교 대학원 2008 국내박사

Due to the rapid progress of biotechnology for massive sequencing capacity over the last ten years, tremendous amount of DNA sequence information has been generated. In order for these data to be processed properly within a relatively short time, it is inevitable to use a sophisticated computer system. Thus it has been widely used for many researchers to identify the genes by using BLAST (Basic Local Alignment Search Tools) service available at National Center for Biotechnology Information (NCBI). The database for BLAST search at NCBI includes non redundant (NR), EST, reference sequence (refseq), genome survey sequence (GSS) and high-throughput genomic sequence (HTGS). In addition, NCBI provides us organism specific BLAST service in case of human, Arabidopsis and mosquito that the entire genome sequencing has been finished. Furthermore, other insect EST and genome databases such as Spodobase (http://bioweb.ensam.inra.fr/spodobase) and Vectorbase (http://www.vectorbase.org) are constructed on the basis of model organisms for the convenience of the users. Up until now, the total number of insect genome projects registered at NCBI is 46. As insect EST and genome project in Korea have been initiated, it has been strongly recommended to build up insect sequnce database for obtaining the rapid and accurate BLAST data. To our knowledge, there has been no report on insect sequence database that is equipped only with insect sequence information at the Web interface in Korea. Based on the concepts described above, the aims of the current study were to (1) construct a pipeline system specialized for genome and EST analysis, and construct a standalone BLAST server with the insect sequence data available from Genbank; (2) conduct two EST projects with Anopheles sinensis and Spodoptera exigua, respectively; and (3) analyze the raw DNA sequence data genereated from these EST projects using the pipeline system developed through this study. Two primary results from Chapter 2 are as follows. First, an efficient analysis system for insect genome and EST was constructed with three sophisticated computer systems. The hardware computer system consists of linux PC server available from Insect Defense Signaling Lab in Chonnam National University, massive and high-throughput sequence analysis system from Inje University, and Edunabi webserver. On the other hand, the softwares for bioinformatic analysis are composed of many sophisticated programs such as BLAST, genescan, PhredPhrap, TGICL, CONSED, FASTA, EMBOSS, SIM4, ClustalW. In doing so, we have finally setup the pipeline system for insect EST and genome project. Second, insect sequence database (ISD) was constructed using the wwwblast package available from NCBI. The diverse advantages of ISD are as follows: (1) It is very easy to conduct BLAST search and to identify the gene of interest; (2) It is much faster than NCBI and has higher chance to get accurate results; (3) ISD is composed of nucleotide sequence, amino acid sequence, mitochondrial genome sequence, and genome sequence. Thus, the users are easy to choose the section they want, and get a data within a very short time; (4) primer3 program was installed so that the users could easy to design their own primer. In conclusion, the system developed in the present study will be very useful for the coming insect EST and genome projects in Korea. In Chapter 3, mosquito EST project was conducted using the pipeline system. Mosquitoes are medical insect that mediates various diseases such as malaria, West Nile virus, Chikungunya virus, Encephalitis virus and dengue virus to human in the world. Recently, malaria has been re-emerged in the demilitarized zone of Korea. In this context, we have been interested in elucidating the molecular interactions between A. sinensis and malaria parasite. In order to identify and obtain the mosquito immune genes, we constructed cDNA library from the human malaria vector, Anopheles sinensis. Then massive and random sequencing was conducted with a sophistigated sequencing system. In addition, we have performed annotation, gene prediction, clustering and KOG analysis. We also constructed webinterface and will be publically open for other users. Furthermore, comparative mapping analysis of As EST sequences with Anopheles gambiae genome data was conducted. Finally, we have obtained various interesting genes such as Toll-like receptor, serpin, dorsal and intergrin through this EST project. We are currently investigating the potential functions of these genes in mosquito midgut in the context of the Plasmodium-mosquito interactions. In Chapter 4, the beet armyworm, Spodoptera exigua was used as a model system. S. exigua is a major agricultural pest that damages to various vegetables and crops in the world. Since it has strong resistance against chemical pesticides, many researchers in the world including Korea have attempted to develop Se-nuclearpolyhedrovirus (NPV) as an alternative biocontrol agent. Recently, we have been involved in elucidating the molecular interactions between S. exigua and Se-NPV. Therefore, we constructed cDNA library with NPV-infected Spodoptera exigua carcass, and also executed massive sequencing with the S. exigua cDNA library. Using the pipleline system specialized for EST analysis we developed in our lab, we have analyzed the raw DNA sequences for annotation, gene prediction, clustering and KOG analysis. From this preliminary study, we have found several genes that may be involved in the SeNPV-Se midgut interactions. Examples of these genes are apolipophorin-III, inhibitor of apoptosis (IAP), transferrin, and 14-3-3 zeta. We are currently elucidating the biological functions of these genes during the battle between SeNPV and Spodoptera exigua. 생명공학기술의 급속한 발달은 genome 연구와 EST project 등을 중심으로 생물학 데이터의 대량화를 가져오고 있으며, 대량화된 정보의 처리를 위해서는 컴퓨터의 이용이 불가피하다. BLAST (Basic Local Alignment Search Tools)는 상동성 (homology)을 이용한 유전자 검색법으로 현재 가장 범용적으로 사용되고 있는 방법이다. National Center for Biotechnology Information (NCBI)에서 BLAST 를 위한 데이터베이스는 non redundant (NR), EST, reference sequence (refseq), genome survey sequence (GSS) 및 high-throughput genomic sequence (HTGS) 등이 기본적으로 제공 되고 있으나 최근 genome 서열이 모두 밝혀진 Human, Arabidopsis, Mosquito 등 30개 종의 생물들의 경우엔 organism specific BLAST 를 제공하고 있으며 또한 NCBI 이외에 현재 운영되고 있는 곤충의 EST 및 genome database는 Spodobase (http://bioweb.ensam.inra.fr/spodobase), Vectorbase (http://www.vectorbase.org)와 같이 각 생물모델 별로 구축되어 운영되고 있다. 이는 각 생물모델을 연구하는 연구자들의 편의성을 도모하는데 그 목적이 있다. 현재까지 NCBI에 등록된 곤충 게놈프로젝트는 총 46개 밖에 없으나 최근 한국에서도 곤충의 EST 및 genome 프로젝트가 시작되면서 신속하고 정확한 BLAST 결과를 얻기위해 곤충의 서열만을 모아서 데이터베이스를 만들 필요성이 증가되고 있으나 아직 곤충의 서열 데이터만을 모아 놓은 서열 데이터베이스를 웹상에 구축한 경우는 없다. 이와 같은 배경에서 설정한 연구목적은 다음과 같다. 첫째, 곤충 expressed sequence tag (EST) 및 곤충 genome분석을 위한 자동화된 pipeline 시스템을 구축하고자 하였으며, 둘째, Genbank에 보고된 곤충 유래 유전자 정보를 수집하여 stand-alone BLAST 서버를 구축하여 관련 연구자들에게 편의를 제공하고자 하였다. 본 연구결과는 다음과 같다. 첫째, 전남대학교에 설치한 리눅스 기반 PC서버, 인제대학교 의과대학에 설치되어진 유전체전용 생물정보 시스템, 함평군 R&D 사업의 일환으로 구축되어진 하나로 internet data center (IDC) 내에 구축되어진 web-server 등에 총 30개 이상의 유전자분석 software (예, BLAST, genescan, PhredPhrap, TGICL, CONSED, FASTA, EMBOSS, SIM4, ClustalW 등) 을 유기적으로 설치하여 보다 빠르고 효율적인 곤충 EST/Genome 분석용 pipeline 시스템을 구축하였다. 본 시스템을 이용할 경우 같은사양의 컴퓨터 시스템에서 실시한 EST 분석에 비해 보다 효율적이고 빠르다는 장점이 있다. 둘째, NCBI의 wwwblast package를 이용하여 insect sequence database (ISD) 서버를 구축하였다. 구축된 ISD 서버는 곤충 연구자들에게 향후 다양한 장점을 제공할 수 있다: (1) BLAST search 통해 ISD에 존재하는 곤충유래 유전자를 사용자가 쉽게 사용할 수 있다. (2) NCBI의 BLAST search 보다 더욱 빠른 속도로 곤충에 관련한 결과만을 연구자들이 얻을 수 있다. (3) ISD는 nucleotide sequence, amino acid sequence, mitochondrial genome sequence, genome sequence로 database를 나누어 놓아 사용자가 원하는 search를 쉽게 수행할 수 있다. (4) primer3 program이 install 되어 있어 사용자가 primer를 design 할 수 있다. 본 연구에서는 이와 같이 개발한 곤충전용 EST/genome pipeline 시스템과 ISD 시스템을 이용하여 모기와 파밤나방의 massive transcriptome sequence 를 annotation 하고 그 결과를 웹인터페이스로 구축하는 EST Project를 수행하였다. 수행결과 본 연구결과를 통하여 구축된 pipeline 시스템은 향후 곤충 EST 및 Genome연구에 많은 도움이 되리라 생각한다. 위에서 개발한 유전자 분석시스템을 이용하여 한국의 말라리아 매개 모기의 EST project를 수행하였다. Anopheles sinensis와 malaria parasite사이에 분자적 상호작용에 관여하는 모기의 면역유전자를 스크리닝하기 위하여, A. sinensis를 이용하여 세계 최초로 cDNA library를 제작하였다. 이후 대용량 시퀀싱을 수행하였고, 확보된 EST database를 annotation, gene prediction, clustering, KOG analysis 등 다양한 방법으로 분석하였다. 분석된 gene을 Anopheles gambiae genome sequence에 comparative mapping을 수행하였다. 또한 web-interface의 구축하였으며, 구축된 web-interface는 추후에 공개할 계획이다. 본 EST를 통해 우리는 toll, serpin, leucine rich repeat protein 그리고 integrin과 같은 다양한 유전자를 확보할 수 있었다. 향후 Plasmodium-mosquito 상호작용에 대한 설명에서 모기의 중장에 존재하는 이러한 유전자들의 잠재적 기능에 대해 조사하고 있다. 또한 파밤나방(Spodoptera exigua)과 핵다각체바이러스(Se-NPV)사이에 분자적 상호작용에 대한 규명하기 위하여, Se-NPV에 감염된 파밤나방의 carcass를 이용하여 cDNA library를 제작하였으며, mass sequencing을 수행하였다. 확보된 EST database를 annotation, gene prediction, clustering, KOG analysis 등 다양한 방법으로 분석하였다. 이 예비연구로부터, 우리는 Se-NPV-Se midgut interaction에 관여하는 몇몇 유전자를 발굴하였다. 이러한 유전자의 예는 apolipophorin-III, inhibitor of apoptosis (IAP), transferrin, and 14-3-3 zeta이다. 향후 항체생산, RNAi, 그리고 생화학적 방법을 이용하여, 이러한 유전자들이 SeNPV와 파밤나방의 상호작용에 어떻게 관여하는 기능을 밝히는데 총력을 기울이고자 한다.