In vitro Propagation of Transgenic Ginsengs Introduced with Ferritin Light Heavy Chain Gene through Single Embryo Culture

Ferritin Light Heavy Chain 유전자가 도입된 인삼형질전환체의 단일배발생을 통한 식물체의 기내증식

  • 윤영상 (공주대학교 식물자원학과) ;
  • 김종학 ((주)바이오피아) ;
  • 김무성 (경희대학교 한방재료가공학과) ;
  • 양덕춘 (경희대학교 한방재료가공학과)
  • Published : 2004.06.01

Abstract

Optimal regeneration conditions of ginseng transformants were studied. It has been known that Ferritin Light Heavy Chain (FLHC) gene remove the several heavy metal by combination, store and transport. To obtain the ginseng tolerant to heavy metal, binary vector was introduced in Agrobacterium by tri-parental mating and then Agrobacterium tumefaciens MP90/FLHC was selected on the AB media and MS media containing kanamycin. Explants were co-cultured with Agrobacterium tumefaciens MP90/FLHC, which contained NPT II as a selectable marker, tadpole ferritin heavy chain (FLHC) gene and human ferritin light chain gene and then a number of embryos were induced. The induced embryo transferred to shooting media consisting of MS medium supplemented with GA 10 mg/L. As a result of examination that induced the normal growth of transfomants, transformants showed the equivalent growth in both root and shoot on the media containing the 1/3 MS.

Ferritin light heavy chain (FLHC) gene는 일부 중금속과 결합, 저장 및 운반하여 무독화 시킬 수 있는 것으로 알려져 있다. Fe 관련 유전자인 FLHC유전자를 식물 발현용 promoter인 35S promoter와 Tnos를 사용하여 식물 형질전환용 vector를 재조합하였다. 식물세포형질전환용 binary vector는 상기 cassette vector가 조립이 매우 양호하며 border sequence를 가지고 있는 pRD400 binary vector를 사용하여 최종적으로 가나마이신 내성 유전자 (NPT II gene)와 tadpole ferritin heavy chain gene 및 human ferritin light chain gene를 함유하고 있는 binary vector를 재조합하였다. Binary vector의 아그로박테리움에 도입은 triparental mating 방법에 의하여 수행하여 AB배지 및 가나마이신 함유 배지에서 disarmed Ti-vector를 가지고 있는 Agrobacterium tumefaciens MP90/FLHC을 선발하였다. FLHC 유전자 도입된 식물형질전환용 binary vector를 이용하여 형질전환방법을 변형하여 많은 embryo를 유도하였으며 유도된 embryo들은 GA 10mg/L가 첨가된 배지에 지상부를 유도하였다. 형질전환체식물체의 정상적인 생장을 유도하기 위해 최적의 배양조건을 조사하였던 바, 비교적 1/3 MS배지에서 뿌리의 생장과 지상부의 생장이 균일하게 생장하는 경향을 보였으며, 뿌리와 줄기가 잘 발달된 약 7cm의 유식물체를 대량으로 증식하여, 모래와 흙이 1:1로 혼합된 토양에 옮겼다.

Keywords

References

  1. Curr Opin Biotechnol v.2 no.6 Genetic marker technology Edwards,A. https://doi.org/10.1016/S0958-1669(05)80113-2
  2. Hypertension v.27 Angiotensin I-converting enzyme gene polymorphism and salt sensitivity in essential hypertension Hiraga,H.;T.Oshima;M.Ishida;G.Kajiyama https://doi.org/10.1161/01.HYP.27.3.569
  3. Rinsho Byori v.41 Detection of methicillin-resistant Staphylococcus aureus using PCR and nonradioactive DNA porbes Huraki,C.K.;K.Taishi;S.Yamashita;Kagawa
  4. J. Bacteriol v.178 Dihydrolipoamide dehydrogenase from the halophilicarchaeon Haloferax volcanii : homology over expression of the cloned gene Jolley,K.A.;E.Rapaport;D.W.Hough;M.J.Danson https://doi.org/10.1128/jb.178.11.3044-3048.1996
  5. The J. of Biol. Chem. v.263 no.21 Purification and Characterization of ferritins from maize, pea, and soybean seeds Laulhere,J.P.;A.M.Lescure;J.F.Briat
  6. Journal of the Korean Society for Horticultural Science v.38 no.2 Changes of Cytokinins and Gibberellin Contents During Low Temperature Storage of Dehisced Ginseng Lee,J.M.;W.S.Kwon
  7. Physiol. Plant. v.15 A revised medium for rapid growth and bioassays with tobacco tissue culture Murashige,T.;F.Skoog https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  8. The J. of Biol Chem v.258 no.18 Ferritin-binding of beryllium and other divalent metal ions Price,D.J.;J.G.Joshi
  9. The J. of Biol. Chem. v.262 no.28 Isolation and Characterization of ferritin from soyabean(Glycine max) Sczekan,S.R.;J.G.Joshi
  10. Zentralbl Gynakol v.122 no.8 Human amniotic fluid transferrin stimulates progesterone production by human trophoblast cells in vitro Streu,A.;U.Jeschke;D.U.Richter;H.Muller;V.Briese;K.Friese https://doi.org/10.1055/s-2000-10607
  11. J. Clin Microbiol v.34 Comparison of PCR detection of mecA with standard susceptibility testing methods to determing methicillin resistance in coagulase-negative staphylococci York,M.K.;L.Gibbs;F.Chehab;G.F.Broohs
  12. The plant cell v.6 Function homologs of fungal metallothionein genes from Arabidopsis Zhou,J.;P.B.Goldsbrough https://doi.org/10.2307/3869965
  13. Plant Cell Report v.1 Isolation of sodium dependent variants from haploid soybean cell culture Zhou,J.P.;E.J.Roth;W.Terzaghi;K.G.Lark https://doi.org/10.1007/BF00269269
  14. 인삼연구보고서(재배분야) 적변삼 원인연구 오승환;박창석;김영인
  15. 고려인삼학회지 v.6 no.2 인삼생육의 최적광량에 관한 연구.제3보. 광도가 다른 조건하에서의 상면피복이 인삼생육에 미치는 영향 이종철;천성기;김요태
  16. 식물유전공학기술개발 최광태;박지창;김갑식;임용표;김남원;양덕춘;안인옥;박성원;이청호;손종현
  17. 식물유전공학기술개발 최광태;박지창;김갑식;임용표;김남원;양덕춘;안인옥;박성원;이청호;손종현;강영희