보고서 정보
주관연구기관 |
국립농업과학원 National Institute of Agricultural Sciences |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2015-03 |
과제시작연도 |
2014 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201500010531 |
과제고유번호 |
1395035293 |
사업명 |
차세대바이오그린21 |
DB 구축일자 |
2015-07-11
|
DOI |
https://doi.org/10.23000/TRKO201500010531 |
초록
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Ⅳ. 연구개발결과
○ 적색, 황색 및 청색 형광고치 생산 형질전환누에(금옥잠)를 개발하였으며 분자육종에 의한 계대사육을 통하여 세대(8세대)를 고정하였다
- 잠125 및 잠140계통별 분리사육 및 우수 형질전환라인 선발
- 우수 형질전환라인과 정상누에 교배를 통한 품종 순계화 추진
- 잠125 및 잠140계통 형질전환누에 게놈 내 형광유전자 도입 위치 확인
○ 녹색형광실크 생산 형질전환누에 역시 14세대 이상 계대 사육함으로써 원종화 유도 및 품종을 육성하였다
- 선발 잠123 및 잠124 계통 우수
Ⅳ. 연구개발결과
○ 적색, 황색 및 청색 형광고치 생산 형질전환누에(금옥잠)를 개발하였으며 분자육종에 의한 계대사육을 통하여 세대(8세대)를 고정하였다
- 잠125 및 잠140계통별 분리사육 및 우수 형질전환라인 선발
- 우수 형질전환라인과 정상누에 교배를 통한 품종 순계화 추진
- 잠125 및 잠140계통 형질전환누에 게놈 내 형광유전자 도입 위치 확인
○ 녹색형광실크 생산 형질전환누에 역시 14세대 이상 계대 사육함으로써 원종화 유도 및 품종을 육성하였다
- 선발 잠123 및 잠124 계통 우수 형질전환누에(제8~13세대)와 정상누에(잠123 및 잠124)의 지속적인 교배에 의한 제14세대 잠123 및 잠124 계통 우수 형질 전환누에 생산 및 품종 원종화 추진
○ 효율적인 형광실크 생산을 위해서 피브로인 H-chain 유전자 프로모터 최대 활성영역을 선발, 효율을 검정함으로써 고효율 피브로인 H-chain 프로모터를 개발하였다.
○ UAS/GAL4 시스템을 리모델링(4×UAS)을 통해 형광실크 대량 생산에 적합한 누에 형질전환 시스템을 개발하였다.
○ 이상의 결과를 바탕으로, 고효율 피브로인 H-chain 프로모터가 도입된 UAS/GAL4 시스템을 구축한 후 이를 이용하여 형질전환누에를 제작하였다. 선발된 형질전환 누에의 계통을 수립하였다(3~4세대)
○ 형광실크을 이용하여 넥타이, 스카프, 블라우스, 전등갓 등 총 4종의 패션소재 시제품을 개발하였으며, 선염디자인 CAD를 이용하여 누에고치와 나비, 꽃 등 자연의 모티브를 활용한 2종의 벽지 디자인 시안을 개발 완료하였음. 또한 패션소재(파티복용) 2종에 대한 디자인 시안을 개발하였다
○ 역할특성, 견뢰도, 인체적합성, 아미노산분석 시험을 통하여 형광실크의 물리화학적 특성 분석하였으며 가공조건 확립하였다
○ 형질전환누에 대량 사육을 통하여 녹색, 청색 및 적색 형광실크를 생산하였다.
○ 형광 누에고치 적합 건조 조건 구명
- 형광 빛을 잃지 않도록 55 ~ 60℃ 저온에서 15시간 열풍 건조
○ 형광 누에고치 적합 제사기술 개발
- 침지액 농도, 감압도, 조사에 따른 60℃이하 자견 및 조사 기술개발
- 감압 자견기 개발로 저온 자견 효율성 향상
Abstract
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We constructed the fibroin H-chain expression system to produce fluorescent protein (EGFP, mKate2, EYFP, BFP, EBFP2, TaqBFP, AmCyan1) in transgenic silkworm cocoon.
Fluorescent cocoon could be made by fusing fluorescent gene to the heavy chain gene and injecting it into a silkworm. The fluorescen
We constructed the fibroin H-chain expression system to produce fluorescent protein (EGFP, mKate2, EYFP, BFP, EBFP2, TaqBFP, AmCyan1) in transgenic silkworm cocoon.
Fluorescent cocoon could be made by fusing fluorescent gene to the heavy chain gene and injecting it into a silkworm. The fluorescent fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the fluorescent gene/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into Baegokjam or Kumokjam, bivoltin silkworm eggs. We obtained broods with EGFP positive eggs. The cocoon was displayed fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.
This study was aimed for development of the more efficient silkworm’s gene transformation system composing a high efficient fibroin heavy chain gene promoter and a modified UAS/GAL4 system. We isolated 8 clones that show stronger signal compared to B. mori cytoplasmic actin gene (BmA3) from B. mori fibroin H-chain 5'-UTR region (-8201~-1). Among the 8 clones, F5R1(-561~-1) was selected the maximum activity region of fibroin H-chain 5'-UTR region. To determine core promoter region, F5R1(-561~-1) was segmented 4 deletion mutants. As result of promoter assay using dual luciferase assay system in the within 4 hrs after oviposition, we found the highest transcription activity core promoter region (-468/-1) in the B. mori fibroin H-chain 5'-UTR region of F5R1-5'-1 gene, which has 6.6 times higher activity than BmA3 promoter. Therefore, we suggest that F5R1-5'-1 core promoter may be used more useful and effectively for making recombinant proteins by transgenic silkworms. We have examine the remodeled UAS/GAL4 system(1~8xUAS) to develop the high expressional silkworm transgenic system in Bm5 cell and silkworm. In the results, the expression level was most highest in case of 4xUAS in Bm5 cell, and also the safety of cytotoxic was observed in the applied transgenic silkworm. Therefore, we suggest that the improved UAS/GAL4 system with a fibroin H-chain core-promoter and a 4xUAS sequences is adequate a transgenic silkworm system for mass production of recombinant proteins.
As pre-study, this study investigated physical and chemical properties of fluorescent silk and tested most felicitous method of degumming to commercialize fluorescent silk fabrics obtained from transgenic silkworm. The result was as follows. Green fluorescent silk is 30 denier on average with large deviations, strength and elongation are each 2.9g/d and 21.7%.
These showed physical properties of fluorescent silk have gradually improved to standard silk although still don`t reached the level of standard silk. There was no difference on Amino acid composition as contrasted with standard silk and skin stimulation test shown as negative that fluorescent gene mutation don`t influence for development the amino acid composition and is appropriate on the human body. The color fastness of washing, dry cleaning, perspiration, rubbing, sunlight are over the 4th grade excepted certain 3rd degree that it seems to be available as textile materials. The measured result of degumming methods by soap alkaline, high pressure, enzyme degumming method, the enzyme degumming kept best fluorescent colors so that is most appropriate. Over 4-ply of fluorescent silk seems to be surely need to complement vulnerability physically of fluorescent silk and to enhance yarn strength. Using fluorescent silk as warp only and combined weaving with standard silk, wool, and others can enhance weaving efficiency. Lastly, multiple pattern designs for blouse, scarf, tie, and dress as clothing materials and for lampshade, wallpaper as interior materials and final products were developed.
The fluorescent proteins are generally denatured by heat treatment and thus lose their color. The normal reeling method includes processing by drying and cooking the cocoons near 100℃ before reeling. Therefore, the usual processing method cannot be used for making colored fluorescent silks. To develop a method that is applicable to producing transgenic silk without color loss, we develop reeling methods adequate for a recombinant fluorescence cocoons. It was found that the fluorescence cocoons keep their native color when dried at temperatures lower than 60℃ for 15 h. Also, a new cooking method to soften the fluorescent cocoons was developed: the cocoons were soaked in a solution of 0.2% sodium carbonate (Na2CO3)/0.1% nonionic surfactant (Triton X100) at 60℃ and then placed under vacuum. The repeated vacuum treatments enabled complete penetration of the solution into the cocoons, and the cocoons were thus homogenously softened and ready for reeling. In this state, the cooked cocoons can be reeled by an automated reeling machine. Our results suggest that Drying and cooking of the cocoons at low temperature enables the subsequent reeling of the colored fluorescent silks by an automatic reeling machine without color loss and can produce silks that can be used for making higher value-added silk materials.
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