들잔디 체세포 배발생 세포로의 DNA 전입을 위한 Electroporation 조건 구명

Electroporation Conditions for DNA Transfer into Somatic Embryogenic Cells of Zoysia japonica

  • 박건환 (경기도 농촌진흥원 원예과) ;
  • 안병준 (단국대학교 농과대학 식물자원학부)
  • 발행 : 1998.01.01

초록

Electroporation을 이용한 형질전환 연구에서 원형질체 대신 체세포 배발생 세포를 이용하여도 DNA가 도입될 수 있음을 이미 보고한 바 있다. 본 연구는 배발생 세포 내로 DNA를 도입하기 위한 electroporation의 최적 조건, 즉 전압과 capacitance 수준, promoter 종류, DNA 농도, 저온처리효과 등을 구명하며, 처리에 따른 DNA의 전입 현상을 이해하고 전기충격후의 생장과 분화 정도를 조사하고자 수행되었다. 들잔디 미숙배를 2,4-D가 2 mg/L 함유된 MS배지에서 배양하여 배발생 캘러스를 유도하였고, 동일 조성의 액체배지에 진탕배양하여 조직 electroporation에 적합한 현탁배양 세포괴를 증식할 수 있었다. 100-400 V의 전압과 10-1980 $\mu\textrm{F}$의 capacitance 수준에서 세포괴를 35S-gusA 조성을 갖는 운반체 DNA와 함께 electroporation 하였을 때, 전반적으로 DNA가 도입되었음을 표지유전자 gusA의 transient 발현을 통하여 확인하였으며, 200-300 V 전압과 330-800 $\mu\textrm{F}$ capacitance 수준이 보다 효과적인 경향을 보였다. 처리시 온도는 큰 영향을 미치지 않았으며, 6 $\mu\textrm{g}$/mL 이상의 DNA 농도에서는 GUS 발현이 양호하였다. 배발생 캘러스 세포주들은 모두 DNA가 도입 되었으나 비 배발생 캘러스 세포주는 11개중 하나에서만 도입이 확인되었다. Electroporation시 전기충격후 20, 40시간 후에 DNA를 첨가하여도 gusA가 발현됨에 따라 전기충격이 세포막의 침투성을 장시간 변화시킴으로써 DNA가 전이될 수 있는 것임을 확인할 수 있었다. GusA의 promoter로 CaMV 35S외에 Actl과 Ubil의 활성을 비교한 바, 35S에 비해 각각 7배, 5배의 활성을 나타내었다. Electroporation 처리후 세포괴의 배양실험에서 100-400 V의 전압과 10-l980 $\mu\textrm{F}$ capacitance의 전 처리 범위에서 캘러스의 지속적인 생장과 함께 식물체 재분화가 일어났다.

We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.

키워드

참고문헌

  1. Biotechnology in Agriculture and Forestry 6: Crop Ⅱ Common bermudagrass [Cynodon dactylon (L.) Pes.]. Ahn BJ;King JW;Huang FH;Bajaj YPS(ed)
  2. Plant Cell Reports v.13 Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts. Asano Y;Ugaki M
  3. Plant Cell Tissue Organ Culture v.12 Electroporation of DNA delivery into plant protoplasts. Bates GW;Piastuch W;Riggs CD;Rabussay D
  4. Annu Rev Plant Physiol Plant Mol Biol v.48 Plant transformation: Problems and strategies for practical application. Birch RG
  5. Plant Science v.81 Optimization of conditions for electroporation and transient expression of foreign genes in sugarcane protoplasts Carls R;Birch RG
  6. Plant Cell Physiol v.33 Transformation of indica (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Chan MS;Lee TM;Chang HH
  7. Plant Cell Mol Biol v.18 Maize polyubiquitin genes: Structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Christensen AH;Sharrock RA;Quail PH
  8. Plant Cell v.4 Transgenic maize plantss by tissue electroporation. DHalluin K;Bonne E;Bossut M;De Beuckeleer M;Leemans J
  9. Virology v.162 Agrobacterium-mediated infectivity of cloned Digitaria streak virus DNA. Donson J;Gunn HV;Woolston CJ;Pinner MS;Boulton MI;Mullineaux PM;Davies JW
  10. Plant Cell Reports v.11 Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. Ha SB;Wu FS;Thome TK
  11. Plant Mol Biol Rep v.5 Assaying chimeric genes in plant: the GUS gene fusion system. Jefferson RA
  12. Plant Cell v.4 Transgenic maize plants by tissue electroporation Kathleen D;Bonne E;Bossut M;Beuckeleer MD;Leemans J
  13. Proc Natl Acad Sci USA v.85 Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Klein TM;Harper EC;Svab Z;Sanford JC;Fromm ME;Maliga P
  14. J Membr Biol v.68 Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. Knight DE;Baker PF
  15. Plant Cell v.2 Isolation of an efficient actin promoter for use in rice transformation. McElroy D;Zhang D;Cao J;Wu R
  16. Mol Gen Gennet v.231 Construction of expression vectors based on the rice actin 1 (Act1)5' region for use in monocot transformation. McElroy D;Blowers A;Jenes B;Wu R
  17. EMBO J v.7 Gene transfer into mouse lyoma cells by electroporation in high electric fields. Neuman E;Schaefer-Ridder M;Wang Y;Hofschneider PH
  18. J Kor Soc Hort Sci v.36 Plant regeneration through somatic embryogenesis in zoysiagrasses (Zoysia spp.) Noh HY;Choi JS;Ahn BJ
  19. Nature v.313 Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Odell JT;Nagy F;Chua N
  20. Plant Cell Physiol v.27 Introduction of functional RNA into plant protoplasts by electroporation. Okada K;Nagata T;Takebe I
  21. Kor J Plant Tissue Culture v.21 DNA delivery into embryogenic cells of zoysiagrass (Zoysia japonica Steud.) and rice (Oryza sativa L.) by electroporation. Park GH;Choi JS;Yun CH;Ahn BJ
  22. Proc Natl Acad Sci USA v.81 Enhancer-dependent expression of human K immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Potter H;Weir L;Leder P
  23. Plant Cell v.1 Uptake and transient expression of chimeric genes in seed-derived embryos. Reinhard T;Gronenborn B;Schell J;Steinbiss HH
  24. Molecular cloning : A laboratory manual. Sambrook J;Fritsch EF;Maniatis T
  25. BioTechniques v.7 Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts. Sauders JA;Smith CR;Kaper JM
  26. Bio/Technology v.3 High efficiency direct gene transfer to plants. Sillito RD;Saul MW;Paszkowski J;Muller M;Potrykus I
  27. Plant Sci v.79 Direct DNA uptake during the imbibition of dry cells. Tissa S;McKersie DB;Kasha KJ;Procunier JD
  28. Seed World Des Plaines v.130 Transformation made easier. van Mellaet H
  29. Plant Cell v.3 Analysis of rice Actl1 5'region activity in transgenic rice plants. Zhang W;McEroy D;Wu R