Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
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Construction of fluorescent red silk using fibroin H-chain expression system

누에 형질전환에 의한 견사선에서의 적색형광단백질 발현

  • Kim, Sung Wan (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Yun, Eun Young (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Choi, Kwang-Ho (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Kim, Seong Ryul (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Park, Seung Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Kang, Seok Woo (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Kwon, O-Yu (Department of Anatomy, College of Medicine, Chungnam National University) ;
  • Goo, Tae Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
  • 김성완 (국립농업과학원 잠사양봉소재과) ;
  • 윤은영 (국립농업과학원 잠사양봉소재과) ;
  • 최광호 (국립농업과학원 잠사양봉소재과) ;
  • 김성렬 (국립농업과학원 잠사양봉소재과) ;
  • 박승원 (국립농업과학원 잠사양봉소재과) ;
  • 강석우 (국립농업과학원 잠사양봉소재과) ;
  • 권오유 (충남대학교 의과대학 해부학과) ;
  • 구태원 (국립농업과학원 잠사양봉소재과)
  • Received : 2012.05.31
  • Accepted : 2012.10.12
  • Published : 2012.12.30
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Abstract

We constructed the fibroin H-chain expression system to produce Discosoma sp. red fluorescent protein variant2 (DsRed2) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing DsRed2 cDNA to the heavy chain gene and injecting it into a silkworm. The DsRed2 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 DsRed2/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 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong red fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the DsRed2 fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

본 연구의 목적은 누에형질전환 기술을 이용하여 적색형광실크를 개발하는 것으로서, 본 실험에서는 피브로인 H-chain의 N-말단과 C-말단을 이용하여 피브로인 재조합 단백질 발현 시스템을 제작하였고, 종결 코돈이 없는 DsRed2 유전자를 위의 발현 시스템에 클로닝하여 적색형광실크를 제작하였다. 누에형질전환체 선발을 위해서는 3xP3 promoter와 EGFP 유전자를 이용하여 선발하였고, 1020개의 누에알에 microinjection 하여 F1 세대에서 6 broods의 형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 EGFP 형광단백질이 발현되는 것을 확인 할 수 있었다. 또한 실크의 피브로인에서 DsRed2 단백질이 발현되는 것을 확인하기 위해, F2세대의 누에형질전환체 중에서 5령 5일 유충을 해부하여 견사선을 형광현미경으로 관찰하였고, 중부와 후부 견사선에서 적색형광단백질이 발현되는 것을 확인 할 수 있었고, F2 세대의 고치에서도 적색형광단백질의 발현을 확인할 수 있었다. 이상의 결과에서 적색형광실크를 생산하는 누에형질전환체가 성공적으로 제작되었음을 확인할 수 있었고 이러한 결과를 토대로 새로운 산업소재로서 실크를 활용할 수 있을 것으로 기대한다.

Keywords

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