• 한국연초학회지
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• 제21권1호
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• pp.57-63
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• 1999

Pokeweed antiviral protein II (PAP-II) encoding cDNA was synthesized by reverse-transcriptase polymerase chain reaction (RT-PCR) from Phytolacca american a leaf. The PAP-II cDNA fragment of 974bp was subcloned to pBluescript II SK- SmaI site and the inserted PAP-II cDNA fragment was sequenced by dideoxy sequencing method. The number of nucleotides of PAP-II cDNA coding region containing start and stop codon was 933bp. To develop a virus-resistant tobacco plant, PAP-II cDNA fragment was inserted to pKGT101B and the insertion of PAP-II cDNA fragment was confirmed by restriction enzyme analysis and colony PCR.

• 한국연초학회지
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• 제23권2호
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• pp.123-132
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• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• 한국연초학회지
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• 제23권2호
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• pp.133-140
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• 2001

PAP-I cDNA was synthesized from total RNA of Phytolacca americana leaves by RT-PCR, and then subcloned to recombinant vector pBluescript II SK-. Using PCR with primers designed in our laboratory, we could get the 9 deletion mutant PAP-I cDNA fragments. The first of the fragments was deleted by 66bp from immature N-terminal and then the rest were deleted by 90bp sequentially. Sequentially deletion mutant PAP-I cDNAs were inserted to pAc55M, on down-stream of gall promoter. Recombinant pAc55M was transformed to yeast cells, psy1 and the cells were spreaded on SC_urn-/glucose plate media. Colonies on SC_ura-/glucose plate were streaked on the same position of SC_ura-/glucose and SC_ura-/galactose plate, and we selected colonies growing on both plates, which carry non-cytotoxic deleted mutant PAP-I cDNA. We selected 4 deletion mutant PAP-I cDNAs which have not cytotoxicity.

• 한국약용작물학회지
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• 제9권2호
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• pp.156-165
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• 2001

현삼의 기내배양에서 낮은 농도의 2,4-D(0.01, 0.1mg/l) 와 TDZ(0.01, 0.1, 2.0mg/l)이 조합처리시 shoot 분화가 좋았으나, 2,4-D의 농도가 높아질수록 TDZ과 조합처리시 shoot 분화가 저조 하였다. 형질전환 확인을 위한 PCR 분석에서 선발표지 유전자로 사용되는 NPT II gene의 확인하였는데 형질전환되지 않은 식물체에서는 나타나지 않는 DNA 절편이 형질전환 식물체에서 나타났으며 kanamycin 50 mg/ l 첨가된 배지에서 선발된 식물체에서 NPT II gene(700bp)이 plant genome 안으로 삽입되었음을 확인하였다. 형질전환 식물체의 항균활성 검정에서는 Asperigillus awamori에 대해 대조구 식물체와 같이 항균성이 없는 것으로 나타났으나 항바이러스성 단백질인 PAP가 도입된 식물체에서는 $IC_{50}$의 값이 Asperigillus awamori에 대해서 각각 $320\;{\mu}g/ml$ 과 $300{\mu}g/ml$, C. herbarum에 대해서도 $IC_{50}$ 값이 $80{\mu}g/ml,\;100{\mu}g/ml$로 높은 항균성을 나타내었다. 형질전환 식물체와 형질전환되지 않은 식물체를 대상으로 SDS-PAGE를 수행하여 본 결과 감염된 형질전환되지 않은 잎과 감염되지 않은 잎에서는 나타나지 않는 30kDa의 분자량을 가지는 새로운 band가 PAP유전자에 의하여 형질전환된 식물체에서 각각 확인되었다. Asperigillus awamori의 경우에 PAP 형질전환체의 단백질을 첨가한 경우 모두 포자가 발아가 억제 되었고 균사생장이 지연되었으며 균사가 생장되더라도 포자와 생장하는 균사가 투명하여졌으며 생장하는 균사의 굵기도 가늘어지는 특성을 나타내었다. 병원균 접종 후 생육조사에 의하면 형질전환 식물체가 초장과 지상부, 지하부의 생체 중에서 모두 형질전환 되지 않은 식물체보다 다소 높게 나타났다. Fusarium에 의한 전형적인 병징인 뿌리썩음 병과 유관속 시들음 증상이 형질전환 되지 않은 식물체에서 병징의 scale은 3.2와 3.0으로 나타나는 반면 형질전환된 식물체에서는 2.0과 2.5으로 비교적 낮게 나타났다.