Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris = Glutathione reductase 유전자의 클로닝에 의한 ozone 抵抗性植物 개발에 관한 연구

국문 초록 (Abstract)

오존을 포함한 광화학 산화물에 대한 저항성을 가지는 목초를 구축하기 위하여 배추의 cytosolic glutathione reductase 유전자를 분리하여 Agrobacterium tumefaciens를 매개로 한 유채 (Brassica napus L ev Chungpoong)로의 형질전환에 관한 실험을 하여 다음과 같은 결과를 얻었다
배추로부터 분리한 glutathione reductase 유전자는 총 길이 1,763bp로서 502개의 아미노산으로 구성된 분자량 54 4kDa의 단백질을 암호화하며, 오존 및 paraquat 처리에 의해 그 발현이 급격히 증가하는 것으로 밝혀졌다 이 유전자를 식물체 형질전환용 벡터인 pBKS1-1의 CaMV 3SS promoter에 subcloning하여 식물체내에서 항상적으로 발현가능한 binary vector, pBKS-GR1을 구축하였다. 구축된 pBKS-GR1을 A grobacterium tumefaciens LBA4404에 도입한 다음, 엽병이 부착된 유채의 자엽과 공배양하여 형질전환을 유도하였다. 유도된 shoots를 kanamycin이 첨가된 재분화배지에서 선발한 다음, 뿌리를 유도하고 완전한 정상식물체로의 발달을 유도하였다 이때 배지중의 AgNO_3 첨가는 shoots의 형성을 크게 증가시키는 것으로 나타났다. Kanamycin이 첨가된 배지에서 선발된 재분화 식물체에 대한 Southern blot analysis 결과, 유채의 genome상에 배추의 glutathione reductase 유전자의 coding sequence (1 8kb)가 정상적으로 도입되었음을 확인하였다

다국어 초록 (Multilingual Abstract)

We have isolated the Brassica compestris cDNA encoding glutathione reductase of 502 amino acid residues with molecular mass of 54 5kDa The deduced amino acid wequences were 92 2%, and 79 5% identical to those of Arabidopsis thaliana, and pea, respectively. As expected, it exhibited a high degree of conservation within the region responsible for the redox reaction and for the binding of GSSG or NADPH The gene was highly inducible by ozone fumigation or by paraquat treatment
This study was conducted to construct of the transgenic plants which are resistant to oxidative stresses including ozone with B. campestris cytosolic glutathione reductase cDNA using the binary vector system of A grobacterium tumefaciens
The 1 8kb B campestris cytosolic GR cDNA was subcloned into the unique Sma Ⅰsite of the plant transformation vector pBKS1-1,downstream of the constitutive CaMV 35S promoter and upstream of the nos termination sequence, in place of the ?dA (GUS) reporter gene. The resulting plant transformation vector, pBKS-GR1, was introduced into A tumefaciens LBA4404 by two cycles of freeze-thaw method. The B napus cotyledonary petioles were transformed by the A grobaterium harboring pBKS-GR1. Transformed shoots were induced and selected on regeneration medium supplemented with kanamycin The shoot formation was increased remarkably by addition of AgNO_3, in MS media. The transgenic plants were analyzed for the presence of the B campestris GR gene by southern blot analysis and it was confirmed that a foreign gene was stably integrated into the genomes of B napus plants.

목차 (Table of Contents)

• Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris
• References
• Transformation of Brassica napus with Glutathione Reductase Gene
• Ⅰ.INTROCDUCTION
• Ⅱ.MATERIALS AND METHODS
• Ⅲ.RESULTS AND DECUSSION
• Ⅳ.SUMMARY
• Ⅴ.LITERATURE CTIED