Korean Journal of Plant Tissue Culture (식물조직배양학회지)

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Expression of Chinese Cabbage Glutathione Reductase Gene in Lettuce (Lactuca sativa L.)

형질전환 상추에서 배추 Glutathione Reductase 유전자의 발현

  • Published : 1998.07.01
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Abstract

Cotyledon explants of lettuce were cocultured with Agrobacterium tumefaciens LBA4404::pBKS-GR1 harboring glutathione reductase(GR) gene in MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 2ip for 48 hr. These explants were transferred to MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 2ip, 50 mg/L kanamycin, and 500 mg/L carbenicillin. After 4 weeks of subculture, kanamycin-resistant shoots were obtained on selection medium. Leaves of putative transformants survived on selection medium containing 100 mg/L kanamycin. Incoporation of the GR gene into lettuce was confirmed by PCR analysis of genomic DNA. Southern blot analysis showed that ECL-labeled GR gene was hybridized to the expected amplified genomic DNA fragment of about 1.8 kb from transgenic lettuce. The presence of mRNA in transgenic lettuce was confirmed by RT-PCR with total RNA of transgenic lettuce. In progeny test of transformants, R$_1$ seeds were resistant to kanamycin (200mg/L) on MS medium.

상추의 자엽 조직을 GR 유전자가 도입된 A. tumefaciens LBA 4404와 2일간 공동배양후, carbenicillin 500mg/L, kanamycin 50mg/L NAA 0.1mg/L와 2iP 1.0 mg/L가 함유된 MS 재분화배지에 옳겨 약 4주후에 kanamycin 저항성 개체를 얻었다. 형질전환된 것으로 추정되는 식물체는 kanamycin 100mg/L가 함유된 MS선발배지에서 생존하였다. PCR 분석결과, GR 유전자가 형질전환체의 게놈상에 삽입되어 있음을 확인하였다. 형질전환체의 Southern blot 분석을 통하여 ECL-labelling된 GR 유전자와 동일한 것으로 판단되는 약 1.8 kb 위치에서 밴드를 확인할 수 있었다. RT-PCR 분석으로 GR 유전자가 전사됨을 확인할 수 있었다. 개화후 이들 개체의 종자를 받아 NPTII 유전자의 발현여부를 조사한 결과 R$_1$ 세대에서도 NPTII 유전자가 발현됨을 확인하였다.

Keywords

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