• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.681-684
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• 2007

An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE3l-bar) and efficiently purified by $Ni^{2+}$ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: $0.01{\mu}g/ml$) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [$4.9{\pm}0.4{\mu}g/g$ of sample (n=6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

• Fisheries and Aquatic Sciences
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• v.19 no.7
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• pp.31.1-31.6
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• 2016

Background: The objective of this study was to develop an efficient selectable marker for transgenic Dunaliella salina. Results: Tests of the sensitivity of D. salina to the antibiotic chloramphenicol and the herbicide Basta$^{(R)}$ showed that cells ($1.0{\times}10^6cells/ml$) treated with 1000 or $1500{\mu}g/ml$ chloramphenicol died in 8 or 6 days, respectively, whereas D. salina cells ($1.0{\times}10^6cells/ml$) treated with 5, 10, 20, or $40{\mu}g/ml$ Basta$^{(R)}$ died in 2 days. Therefore, D. salina is more sensitive to Basta$^{(R)}$ than to chloramphenicol. To examine the possibility of using the phosphinothricin N-acetyltransferase (pat) gene as a selectable marker gene, we introduced the pat genes into D. salina with particle bombardment system under the condition of helium pressure of 900 psi from a distance of 3 cm. PCR analysis confirmed that the gene was stably inserted into the cells and that the cells survived in $5{\mu}g/ml$ Basta$^{(R)}$, the medium used to select the transformed cells. Conclusions: The findings of this study suggest that the pat gene can be used as an efficient selectable marker when producing transgenic D. salina.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2002.11b
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• pp.30-30
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• 2002

꿩의비름 (Sedum erythrostichum)은 매우 우수한 지피식물이며 건조에 강한 대표적 식물로 바위정원 (rock garden)을 가꾸는데 있어서 중요한 수종으로 이용되며, 유럽등지에서는 지붕에 식재하기도 하며 최근에는 빌딩옥상녹화의 대표적 수종으로 식재되고 있다. 또한 한방에서는 경천이라 불리우기도 하는데 피부상처 치유 및 미백효과가 탁월하다고 알려져 있다. 본 연구에서는 Agrobacterium을 매개로한 꿩의비름의 형질전환 시스템을 개발하고 아울러 phosphinothricin-N-acetyltransferase (PAT) 유전자를 도입하여 제초제 저항 식물을 개발하고자 수행되었다. 꿩의비름 잎을 Agrobacterium에 담근후 0.5 mg/l NAA와 2 mg/1 BA가 첨가된 MS 배지에 3일간 공동배앙 하였다. 그 후 300 mg/1 cefotaxime이 첨가된 같은 배지에 옮겨 계대하면서 Agrobacterium을 제거하였다. 약 3주후에 잎 절편으로 부터 직접적으로 부정아가 형성되기 시작 하였는데 이 시기부터 잎 절편을 25 mg/1 kanamycin이 첨가된 선발배지에 옮겨 주었다. 이 결과 배양된 잎 절편 절편 중 3.75%에서 kanamycin에 저항하는 부정아를 얻을 수 있었다. 형질전환체는 X-gluc 반응, PCR, Southern, Nothern analysis를 통하여 확인하였다. 약 94%의 형질전환 식물체는 성공적으로 토양에 옮길 수 있었으며 약 3개월후에 꽃을 피웠다. 형질전환체는 제초제인 Basta ($^{(R)}$ phosphinothricine at 200 mg/1)를 살포하여 주었을 경우 생존함을 확인 하였다.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.281-287
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• 1999

Codonopsis lanceolata ("Deoduck" in Korea) is a perennial herb, and belongs to family, Campanulaceae. Its taproot is used a good source of a wild vegetable as well as an herbaceous medicine. In this study, to develop a bialaphos-resistant transgenic Codonopsis, seed germination mechanism and somatic embryogenesis of the plant were investigated, and Agrobacterium-mediated transformation with bar gene encoding phosphinothricin acetyltransferase (PAT) was performed. Attempt were made to regenerate plant from cells via somatic embryogenesis. When the cotyledons, nodes and leaf disks were cultured on MS medium containing 2,4-D and zeatin, embryogenic calli were induced. Upon transferring the somatic embryos to N6 solid medium without plant growth regulators, they developed into plantlets under continuous illumination. All plants were dead on MS basal medium containing 10 mg/L phosphinothricin (PPT) and Basta, respectively. The explants did not produce calli in the medium containing 200 mg/L kanamycin. The explants were cocultured with Agrobacterium tumefaciens for 2 days, and transformants were selected in MS basal medium containing 1.0 mg/L 2,4-D, 100 mg/L kanamycin and 500 mg/L carbenicillin. After the selection, embryogenic calli were induced and then somatic embryos were produced by subsequent subculturing. The somatic embryos were germiated on N6 basal medium containing 200 mg/L kanamycin and 500 mg/L carbenicillin. PCR analysis showed that nptII and bar genes were introduced in the Deoduck transformants. After the confirmation of bar gene expression in RNA and protein level, the transgenic Deoduck will be used to study the genetics of filial generation with the herbicide control gene, bar.gene, bar.