초록 ▼

본 연구개발은 해양용 LMO의 개발, 생산, 유통 및 관리에 필요한 LMO 평가, 심사 및 검사 기술을 확보함으로써 LMO로 인한 해양생태계 위해성 저감 및 대응방안을 마련, LMO의 잠재적 위해성으로부터 국내 해양생태계를 보존하기 위함이다. LMO 위해성 관리 및 평가 기반 기술 개발을 위하여 기초 생물 특성 등의 생태학적 선행 연구 조사를 바탕으로 해산어류(Oryzias dancena), 해양식물(Undaria pinnatifida, Saccharina japonica, Dunaliella salina) 및 해양미생물(Halob

본 연구개발은 해양용 LMO의 개발, 생산, 유통 및 관리에 필요한 LMO 평가, 심사 및 검사 기술을 확보함으로써 LMO로 인한 해양생태계 위해성 저감 및 대응방안을 마련, LMO의 잠재적 위해성으로부터 국내 해양생태계를 보존하기 위함이다. LMO 위해성 관리 및 평가 기반 기술 개발을 위하여 기초 생물 특성 등의 생태학적 선행 연구 조사를 바탕으로 해산어류(Oryzias dancena), 해양식물(Undaria pinnatifida, Saccharina japonica, Dunaliella salina) 및 해양미생물(Halobacterim sp.) 모델의 숙주종을 선정, 평가 모델 시료 개발 및 계통을 확립하였다. LM 모델과 모의생태계를 기반으로 생식학적 또는 비생식학적 경로 등을 통한 LM 어류로 인한 생태학적 전이 및 위해 가능성을 평가하였다. 또한 해양환경 협의심사를 위한 타용도(농업용 및 수산용) LMO의 해양생태계 전이 가능성을 평가하여 이를 바탕으로 전파 경로 및 양상 탐색, 해양생물체내 검출 빈도 등을 연구하였고, LM 어류 위해성 평가 지표 개선을 위한 분석 기술의 검증 및 표준화를 위한 LM 어류의 다량 신속 분석법 및 Kit 등을 개발하였다. 이들 결과를 바탕으로 LM 어류 위해성 평가 표준 매뉴얼 등을 개발하였고 향후 해양생태계 LMO 안전관리 정책 지원 및 제도 개선에 이용될 것이다.

Abstract ▼

IV. Results

1.Biological characteristics of risk assessment model organisms

A. Ecological, phylogenetic and population genetics analyses
To develop risk assessment procedures we selected marine fish, plants and microbes, based on the results of an earlier study of their biological c

IV. Results

1.Biological characteristics of risk assessment model organisms

A. Ecological, phylogenetic and population genetics analyses
To develop risk assessment procedures we selected marine fish, plants and microbes, based on the results of an earlier study of their biological characteristics and habitats. The marine euryhaline medaka Oryzias dancena was selected as a model marine fish. The mitochondrial cytochrome b gene (cyt-b) and the nuclear recombination activating gene 1 (Rag 1) of three species of the genus Oryzias (O. dancena, O. sinensis and O. javanicus) were identified, and microsatellite markers were developed for phylogenetic and population genetic analyses. The results of analysis of the full-length mitochondrial genome (mitogenome) sequences of the three Oryzias species showed that they have high homology with other vertebrates. As model marine plants the brown alga Undaria pinnatifida, the kelp Saccharina japonica, and halophilic green algae Dunaliella salina and D. tertiolecta were selected, and their mitochondrial cytochrome c oxidase III (Cox3) genes and nuclear internal transcribed spacer (ITS) regions were determined for phylogenetic analysis. Although the S. japonica group is monophyletic, U. pinnatifida is closely related to U. undarioides and U. peterseniana. Phylogenetic analysis for Dunaliella sp. was performed using the ITS region, and showed it was associated with a clade including D. salina and D. tertiolecta. Halobacterium sp. was selected as a model marine microbe, because of its capacity for adaptation to high salinity.

B. Analysis of cytogenetic and molecular genetic characteristics
Cytogenetic analysis was performed to assess the biological properties of O. dancena through the karyotyping, erythrocyte area, frequency distribution of erythrocyte nucleolar organizer regions (NORs) and cellular DNA content. The genome size (130 Mb) of D. salina and the complete genome sequence of Halobacterium sp. NRC-1 were obtained from publicly available information. To analyze the molecular characteristics of O. dancena, genomic and cDNA libraries were constructed and total RNA was prepared for isolation of target genes. In addition, the tolerance of O. dancena to various salinity conditions was assessed, and immune system-related genes and genes expressed in response to pollution were analyzed.

C. Physiological and pathological characteristics
To determine the optimum multiple and daily spawning conditions for O. dancena we investigated egg development, reproductive behavior, osteological development, sex differentiation and gonad development. In addition, the osmoregulatory capabilities of adults and larvae were studied. O. dancena was able to spawn eggs under a wide range of salinity conditions (0-70 ppt), and there was no adverse effect on viability following direct transfer from fresh water (0 ppt) to 40 ppt salinity, or from 70 ppt to 0 ppt. At 10 ppt salinity O. dancena showed a strong immune response to Edwardsiella tarda relative to its response under other salinity conditions. Indoor culture techniques were developed for U. pinnatifida and S. japonica, and indoor culture of the microalgae and microbe was performed using previously reported methods.

2. Development of model organisms for risk assessment for marine LMOs

A. Optimization of conditions for genetic manipulation
The optimal conditions for gene manipulation were developed for the model organisms. The optimal genetic manipulation conditions for O. dancena were established based on photoperiod, the female:male ratio, egg quality improvement, and the delay of eggshell calcification. For the model marine plant and microbe, particle bombardment, electroporation and glass bead methods were used to establish a preconditioning process for gene transfer.

B. Induction of transgenic fish

(1) Construction of transgenes and protein expression assay
The endogenous inducible promoters of O. dancena were obtained through gene cloning, and analysis of sequences, gene structure and transcriptional regulatory factors. Based on these results we constructed expression vectors for evaluation of induction of fluorescent protein expression and promoter activities (podb-actRFP, podmlc2-1RFP, podmlc2-2RFP), for assessment of the marine environment (podChoH-1RFP, podChoH-2RFP, podChgL-1RFP, podMTpRRFP, podER-1RFP, podER-2RFP), and for induction of fast growth (podMTpRGH(+), podMTpRGH(-), podb-actGH(+), podb-actGH(-)). In total, 15,000 eggs (1000 eggs/group) were micro-injected; the mean percent hatching and survival rates of early larvae were approximately 15% - 20% and 80%, respectively. All the founder transgenic fish had fluorescent bodies, although the sites of fluorescent protein expression and the fluorescence intensity were variable among them.

(2) Development of model fishes for risk assessment for LMOs
• The strains showing uniform expression of fluorescence signals throughout the body (cytoskeletal b actin) throughout its life cycle was selected for identification of the transferred gene.
• The strain showing significant expression of fluorescence signals at specific sites, particularly skeletal tissue (myosin light chain 2) was selected.
• The strain having strong fluorescence protein expression in particular external environments (choriogenin H/L isoform), and having the ability to inhabit a wide range of salinities (from freshwater to seawater) was selected.
• There was no continuous overexpression of growth hormone in O. dancena. However, their early growth was accelerated and they showed high levels of abnormality.

C. Induction of transgenic marine plants and microbes

(1) Construction of transgenes and protein expression assay
Efficient methods for transferring foreign genes into marine plants and microbes have not yet been established. In this study, reporter constructs were used to generate transgenic plants and microbes. For U. pinnatifida and S. japonica the optimal conditions (e.g., particle distance and pressure) for particle bombardment were developed using two fluorescent protein expression vectors and one b-galactosidase immunohistochemistry vector; the resulting transgenic algae expressed fluorescent protein in the thallus tissues. To transfer DNA into marine microalgal cells, glass bead methods were used with Chlamydomonas， and gene expression was identified though PCR analysis. The methods for the transfer of foreign genes into microbes were also developed.

(2) Development of model plants and microbes for risk assessment for LMOs
Inspection testing procedures were established during this study, and an inspection manual for U. pinnatifida and S. japonica was prepared. The field of transgenic microalgae is still in its infancy. However, fundamental biological processes have been reported in numerous studies. In this study we established a stable transgenic line to improve the efficiency of gene transfer. With respect to transgenic microbes, plasmid DNA containing a chimerical promoter was injected into Escherichia coli and Edwardsiella tarda for recombinant protein production.

3. Development of techniques for marine LMO genome analysis

A. Standardization of risk assessment for genes transferred into transgenic fish

Stable transgenic germ lines carrying the red fluorescence protein (RFP) gene driven by cytoskeletal b-actin, and fast skeletal myosin light chain-2 (mlc2f) gene promoters, were established in O. dancena. The transgenic lines contained various transgene copy numbers, and the transgenic founders displayed mosaic and/or ectopic expression of the RFP signal. Stable transmission of the transgenic locus and uniform expression of the RFP signal from the F1 generation to all subsequent generations was identified by dot blot and real-time PCR analyses.

B. Standardization of the technique for detection of transgenes in transgenic fish

Quantitative real-time RT-PCR showed that there was little difference in the expression patterns of transgenes under the regulation of b-actin and MLC2 promoters. Based on the real-time RT-PCR assay, the transgene and endogenous genes could be found in several tissues, and the transgene and endogenous gene RFP levels were stable in the transgenic fish muscles through several generations. Western blot analysis showed that the overall pattern of the tissue distribution of transgene transcripts resembled that of the endogenous gene.

4. Ecological LMO risk assessment based on reproductive or non-reproductive physiological processes

A. Risk assessment for transgene transfer based on reproductive physiological process

Risk assessment for transgene transfer by intraspecific or interspecific breeding was examined. Embryonic and larval stages from intraspecific breeding showed physiological abnormalities including lowered growth rate, delayed sexual maturation and significant early mortality. Analysis of interspecific breeding showed the potential for hybrid formation and stable germ-line transmission. Abnormalities in embryonic and larval stages were observed, but no unusual abnormality was caused by transgenes relative to non-transgenic lines.

B. Risk assessment for transgene transfer by non-reproductive physiological process

• An assessment of the risk of transgene transfer by predation was performed. The transgene was detected in whole organs (or tissues) of predatory fish, and in their feces. Although endogenous genes of O. dancena were also found, the frequency was significantly lower than that of the transgene.
• Assessment of the risk transgene transfer in transgenic fish feces showed that short transgene units, antibiotic resistance genes and/or high copy endogenous genes were excreted in fish feces.
• In a short-term experimental study of materials, including feces, from transgenic fish reared in fish tanks, the transgene was detected in all tanks, while the endogenous gene was detected at low frequency in materials from the bottom of the tanks.
• The frequency of detection of biogenic bacteria in transgenic fish was investigated. Expression of fluorescent protein in the recombinant bacteria was used to distinguish them from other aquatic bacterial infection; no transgene transfer by bacterial infection was detected.

C. Assessment of risk of transgene transfer by transgenic fish for the marine ecosystem

• An environmental simulation was performed for 6 weeks to estimate the possibility of transfer of transgenes from transgenic fish and GM crops to invertebrates. The transgenic fish transgene was detected in predators after two weeks, while the endogenous genes were not found. In O. dancena the possibility of transgene transfer by non-reproductive physiological process was identified. A GM crop transgene was also detected at relatively high frequency.
• An ecosystem simulation modeled on the Yeonhwari (Kijanggun, Busan, Korea) was performed for 44 weeks to estimate the transfer of transgenic fish transgenes expressing RFP and commercial feed including GM crops. The transgenic fish transgene was detected in the internal tissues of green spotted puffer fish (Tetraodon nigroviridis) fed the transgenic fish, but the frequency was very low. In contrast, the transgenes of the GM crops were detected at high frequency.

5. Assessment of the risk of LMOs for the marine ecosystem

A. Risk assessment for fisheries LMOs

Analysis and detection techniques were developed to prepare for the adventitious presence or illegal distribution of transgenic zebrafish expressing fluorescent proteins. These fish had stable lines that expressed in the germ line for many generations, and the fluorescent proteins were detected in all tissues. However, the potential for adventitious presence of transgenic zebrafish is greater because in poor health they are not easily distinguishable from non-transgenic fish. Thus, in this study we optimized multiplex PCR conditions for the detection of endogenous and transgenes of transgenic zebrafish.

B. Assessment of the risk of agricultural LMOs for the marine ecosystem

The effect of GM crops (soybean and corn) used in feed for cultured fish (flatfish, jacopever) was examined. There was no statistically significant difference in weight, body composition, food coefficient, and survival rate among all experiment groups except for jacopever fed GM corn (20%). The effects of GM feeds on the survival, growth and reproductive rates of O. dancena were also investigated, and somatic and reproductive cell changes and transgene transfer in feces were identified. In addition, the transfer of transgenes from GM crops to invertebrates inhabiting areas near the fish farms was confirmed.

6. Monitoring transfer of transgenes from GM crops to the marine environment

A. Investigation of the impacts of fish feed and raw materials on the marine environment

• The ingredients of fish feed (dehulled soybean cake, soybean cake, corn gluten meal, corn, rice bran, wheat flour, distillers dried grains, and wheat) used by a food company were examined for the presence of GM proteins and/or GM genes. GM proteins and/or GM genes were detected in rice bran, wheat flour and wheat, and are banned for import into Korea.
• The inclusion of GM crop products in halibut feed from Europe (Netherlands and Denmark), Japan (4-6 times each year) and Korea (5-7 times each year) was investigated, and evidence of the presence of gene structures from GM crops was found. In addition, most feeds lacked labeling indicating that they contained products derived from GM crops, amongst which were halibut feed but also various domestic feeds (eel, abalone, sea bass, trout, and tilapia).

B. Sea monitoring for GM genes

• In 2011 the frequency of detection of GM genes among season and species was investigated in 11 areas of Jeju Island. Transgenes from GM crops were detected in all areas without reference to adjacent or not adjacent fish farms, and there was no significant difference among seasons and species.
• In 2012 the frequency of detection of GM genes was investigated among season, feeds, and bottom materials in 8 areas of the southern coast of Korea (Geoje, Namhae, Yeosu, and Wando). Transgenes from GM crops were detected in all areas without reference to adjacent or not adjacent fish farms, and there was no significant difference among seasons, feed, and bottom materials.
• In 2013 the frequency of detection of GM genes among season, feeds, and bottom materials was investigated in 8 areas (Yangyang, Samcheok, Goseong, Gijang, Dangjin, Taean, Boryeong, and Seocheon) of the eastern and west coasts of Korea. The results were similar to those found at Jeju Island and along the southern coast.
• The PCR assay for transgenes from GM crops detected partial transgene gene structures, using species-specific primers.
• Transgenes from GM crops were detected in Namwon (Jeju Island) and Dapo-ri (Geoje), but there was no significant difference among seasons and adjacent or not adjacent fish farms.

C. Monitoring for transgenes from GM crops around fish cage culture farms and in imported marine products

GM genes were detected in invertebrates and bottom materials sampled adjacent to a fish cage culture farm, and GM genes were also identified in marine products available for sale.

7. Development of the mass and rapid analysis techniques and an inspection kit

• For detection of selected genes, structural genes, promoters, and junction regions, primers were designed and PCR conditions were optimized. Based on these conditions a multiplex PCR technique was developed for detection of more than two transgenes.
• Mass and rapid analysis (15 min) techniques were developed for LMOs (all tissues and organs of fish, artemia, microalgae, and invertebrates).
• An inspection kit for testing transgenic fish was developed to asses adventitious presence, and an instruction manual was prepared. This inspection kit enables rapid analysis (80 min) and is easy to use without specialized equipment.