초록 ▼

○ GS-FLX system을 이용하여 해양공생생물 총 18종의 시료에 대한 발현 유전체 분석을 완료 하였으며, 해독된 전체 유전체 양은 약 5.6Gb로 적조유발종의 데이터베이스와 웹 기반 분석 pipeline을 세계 최초로 구축하였다 (http://genebank.kongju.ac.kr)
○ 비교유전체학적 방법을 이용하여 바다민달팽이와 깃털말간의 유전자 전달과정을 연구하였으며, Actin 유전자와 광합성관련 유전자의 수평적 유전자 전달을 확인하였다.
○ 혼합영양성 섬모류, 편모류, 와편모조류에 대한 단종 배양체제와 먹이

○ GS-FLX system을 이용하여 해양공생생물 총 18종의 시료에 대한 발현 유전체 분석을 완료 하였으며, 해독된 전체 유전체 양은 약 5.6Gb로 적조유발종의 데이터베이스와 웹 기반 분석 pipeline을 세계 최초로 구축하였다 (http://genebank.kongju.ac.kr)
○ 비교유전체학적 방법을 이용하여 바다민달팽이와 깃털말간의 유전자 전달과정을 연구하였으며, Actin 유전자와 광합성관련 유전자의 수평적 유전자 전달을 확인하였다.
○ 혼합영양성 섬모류, 편모류, 와편모조류에 대한 단종 배양체제와 먹이 배양체제를 구축하였으며, 이들에 대한 대량 발현 유전체 해독을 실시하였다.
○ 혼합영양성 편모류의 먹이생물을 기초로 유연관계를 나타내는 Trophic tree 작성방법을 세계최초로 개발하였다.
○ 혼합영양성 편모류 대표종을 대상으로 포식된 먹이로부터 포식자로의 유전자 전달 및 기능 작동 여부 규명하였다.
○ 전자현미경을 이용하여 도입된 색소체의 안정성과 형태적, 생리적 특성을 규명 하였으며, 도입된 색소체의 역할을 제시 하였다.
○ 생태학적, 생리학적, 세포학적 및 진화적으로 매우 특이한해양 생물 종인 혼합영양성 섬모류 Myrionecta rubra (= M. rubrum)를 대상으로, 색소체 제공 원인먹이생물의 다양성과 훔친엽록체 조절에 관여하는 전달유전체에 관한 배양실험 연구를 수행하였다.
○ 와편모조류 기능성 유전자(HSP70, HSP90, CYC, KatG, CRT, GST) 클로닝, 게놈구조, 유전자전달 및 real-time RT-PCR 발현양상 조사하였다.

Abstract ▼

IV. Results
○ The transcriptome analysis of 18 marine symbiotic organism were completed. Total 30.4Gb of transcriptomes were obtained using GS-FLX system (NGS sequencing) as well as Highseq and submitted to Genebank in Kongju National University (http://genebank.kongju.ac.kr)
○ Sea slugs (7 sp

IV. Results
○ The transcriptome analysis of 18 marine symbiotic organism were completed. Total 30.4Gb of transcriptomes were obtained using GS-FLX system (NGS sequencing) as well as Highseq and submitted to Genebank in Kongju National University (http://genebank.kongju.ac.kr)
○ Sea slugs (7 species) in Korea were collected and identified based on their morphology and gene sequencing.
○ Most of the identified sea slugs showed diet specificity. They mainly feed on marine coenocytic green algae Bryopsis spp. and Chaetomorpha moniligera. Some species feed on red algae Antithamnion densum, A. glanduliferum, and Aglaothamnion byssoides.
○ Chlorophyll a fluorometry showed that the photosynthetic activity of kleptoplasts in the sea slugs could be maintained for a long time under low-light condition.
○ The chloroplast-containing digestive cells were located beneath the layer of vacuolated cells. The digestive cells contained 10-15 chloroplasts and several nuclei. Most chloroplasts were intact and contained aligned thylakoids, pyrenoid surrounded with starch grains, and plastoglobule lipid bodies.
○ Total 1.2Gb of transcriptome was obtained from sea slugs, Elysia nigrocapitata, Elysia atroviridis, Placida dendritica, and marine green alga, Bryopsis plumosa using Next Generation Sequencing (NGS, GS-FLX).
○ Actin gene as a possible transferred gene between alga and sea slug was isolated by comparing of two transcriptomes.
○ We compared similarities of intercellular organisms between predator and prey species by using TEM, and found that when fed, intercellular organisms maintained within predator’s inner cellular part. Then, we developed specific primer of prey species to discover maintanance of prey’s genes when fed by predator and discovered that gene of prey maintained within predator for several days to weeks, or transferred to predator’s gene without getting digested.
○ When we investigated transcriptome of 5 marine mixotrophic dinoflagellate species to reveal wether prey genes actually transferred to predator’s gene and found that not only the genes of prey that were provided previously, but also the genes of potential or prey species that were not provided from the result.
○ We investigated exact species that genes were found within transcriptome data of 4 predator species and confirmed that the genes of prey species and possible species are transferred to predator species gene and are expressed and have abilities to function as predator’s gene after it is transferred.
○ We established 24 mixotrophic flagellates (11 species already obtained. Another 13 newly established species in Korean waters collected from 25 stations along the eastern, western, and southern coasts).
○ We found the kind of prey which these 24 mixotrophic flagellates were able to feed on using the light microscopy, epifluorescence microscopy, and transmission electron microscopy. Furthermore, we analyzed phylogenetic tree using the sequences of rDNA of the mixotrophic flagellates. In addition, we developed the trophic trees based on the kind of prey and genetical characterizations of species belonging to 5 major orders (Dinophysiales, Gymnodiniales, Peridiniales, Phytodiniales, Prorocentrales).
○ We established successfully two strains of the mixotrophic ciliate Myrionecta rubra (=Mesodinium rubrum) and its cryptophyte prey in laboratory culture for use as model organisms to investigate plastid acquisition and endosymbiotic gene transfer.
○ We demonstrated that population growth and plastid type of Myrionecta rubra depend on the available preys of cryptophyte through molecular techniques and feeding experiments. We observed initial mechanism for plastid acquisition based on TEM ultra sections.
○ Determination of physiological growth responses of notorious marine armoured and unarmored dinoflagellates (Amphidinium, Gyrodinium impudicum, Prorocentrum minimum)
○ Determination of physiological photosynthetic efficiency and median effective concentration (EC50) of the test dinoflagelaltes exposed to environmental stressors
○ Measure nuclear genome size of local dinoflagelalte Prorocentrum minimum (ca. 9.1 Gb)
○ Construction of cDNA from mRNA isolated from various culture conditions (temperature, salinity, contaminants) (602 cDNA)
○ Determined 274,250,000 ESTs and 30.4 Gb DNA seuqnces from test dinoflagelates using NGS techniques (GS-FLX Titanium, HiSeq2000), and their EST data characterization
○ Discovery unique gene ORF 50,321 from dinoflagellate EST data, and gene annotation of 30,923 from Prorocentrum minimum by using NCBI BLAST-X searches
○ Gene ontology (GO) analyses with donoflagellate EST data, and dtermined each gene
○ Gene cloning of housekeeping genes (Actin, Tubulin, cyclophilin, GAPDH) by using 5‘-, 3’-RACE techniques
○ Molecular gene cloning of functional genes (HSP70, HSP90, CYC, KatG, CRT, GST), genomic structure, gene transfer, phylogenetic relationships, and gene expression level using real-time RT PCR
○ Based on individual genes of dinoflagellates, characterized complicate dinoflagellate genomic structure (intron, exon, gene copy, tandem repeat multicopy, mRNA editing)
○ Analyzed activities of antioxidant enzymes catalase, reduced glutathione of Prorocentrum minimum exposed to environmental pollutants
○ Isolation cellular organelles (chloroplast, mitochondria, plastid) from armoured and unarmored dinoflagellates, and determination of their genomes
○ Characterization of dinoflagellate Prorocnetrum minimum mitochondria genome, and unusual gene arrangements
○ Discovery of dinoflagellate mitochondrial CO2 gene (CO2a, CO2b segregation and intron) sequences and nuclear lateral gene transfer (LGT) ○ Chloroplast gene cloning light and dark reaction in photosynthesis (692 numbers)
○ Determination of chloroplast gene psbA and atpB minicircle, and rbcL gene transfer in Prorocentrum minimum
○ Dinoflagerllate Prorocentrum minimum genome sequencing using GS-FLX Titanium, and its genome characterization (ORF prediction, chloroplast, mitochondria gene, symbiont gene)
○ Large-scale genome sequencing of Prorocentrum minimum uisng illumina Hiseq2000 (30.4 Gb sequences) and discovery of unique gene and genomic characters (polyploid, repetitive, symbionts)
○ Characterization of dinoflagellate as Mesokaryote in terms of marcroevoluion from prokaryote to eukaryotes

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 9CONTENTS ... 15목차 ... 16제 1 장 연구개발과제의 개요 ... 17 제 1 절 연구개발의 목적 ... 17 제 2 절 연구개발의 필요성 ... 18 제 3 절 세부과제 구성 및 연구개발의 범위 ... 23제 2 장 국내외 기술개발 현황 ... 25제 3 장 연구개발수행 내용 및 결과 ... 31 제 1 절 발현 유전체 해독 및 유전체 은행 구축 ... 31 제 2 절 세부과제별 주요 연구내용 ... 37제 4 장 목표달성도 및 관련분야에의 기여도 ... 54 제 1 절 연구개발 최종목표, 연차별 연구개발 목표 및 내용 ... 54 제 2 절 최종 연구개발 목표 달성도 및 관련분야 기여도 ... 58제 5 장 연구개발결과의 활용계획 ... 60제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 62제 7 장 참고문헌 ... 64끝페이지 ... 67