Frankia sp. strain SNU 014201의 nif-H, D, K, 유전자 클로닝

권석윤,강명수,안정선 한국미생물학회 1992 미생물학회지 Vol.30 No.1

물오리나무의 뿌리혹에서 분기한 Frankia sp. SNU 014201 공생균주의 게놈내에 13.5 kb의 EcoRI, 18.0 kb 의 BamHI, 10.5 kb의 Bg/II, 4.5 kb의 KpnI 절편에 nif-H, D 유전자가 존재함을 확인하였다. 람다 파아지 EMBI3-BamHI arm을 사용하여 제조한 genomic library 에서 nif유전자를 포함하고 있는 14개의 재조합 파아지 클론을 선별하였다. 이들 중 Ahnif-12번 클론은 nif 유전자를 포함하고 있는 18kb 의 삽입 DNA 를 가지고 있었으며, 이중 7.9 kb 의 BamHI 절편내에 nif-H. D. K가 3.6kb 의 HindIII/KpnI 절편내에 nif D 의 일부와 H 가 위치하고 있었다. 따라서 이등 절편을 각각 subcloning 하고 제한효소 지도를 작성한 결과, Frankia sp. SNU 01420의 nif-H. D. K 유전자는 6.5 kb 의 Hind III/Bam HI 절편과 5.2 kb Sal/IBamHI 절편내에 연속 배열하고 있다. nif (nitrogen fixation)-H.D, K genes of Frankia sp. SNU 014201. a symbiotic strain isolated from root nodule of Alnus hirsura, were found to be located in the genome on 13.5 kb of EcoRI, 18.0 kb of BamHI, 10.5 kb of BglII and 4.5 kb of KpnI fragments. Using EMBL-3 BamHI arms of bacteriophage lambda. the genomic library was constructed. from which fourteen recombinant phage nif-clones were selected. Among them, Ahnif-I2 had insert DNA of 18 kb, in which 7.9 kb of BamHl fragment contained nif-H, D, K and 3.6 kb of HindlIl/KpnI had nif-H and partial -D. Therefore, the 7.9 kb and 3.6 kb fragments were subcloned and partial restriction maps were constructed. As the results, nif-F1, D.K genes were found to be located continuously on the 6.5 kb of HindII/BamHI and 5.2 kb of SalIIBamHI fragment in the genome of Frankia sp. SNU 014201.

엽록체 항산화기구 대사조절에 의한 환경스트레스 내성 식물

권석윤,곽상수,임순,이행순,이영표 한국식물생명공학회 2005 JOURNAL OF PLANT BIOTECHNOLOGY Vol.32 No.3

엽록체 항산화기구 대사조절에 의한 환경스트레스 내성 식물

권석윤,이영표,임순,이행순,곽상수,Kwon Suk-Yoon,Lee Young-Pyo,Lim Soon,Lee Haeng-Soon,Kwak Sang-Soo 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.3

Injury caused by reactive oxygen species (ROS), known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Chloroplasts are specially sensitive to damage by ROS because electrons that escape from the photosynthetic electron transfer system are able to react with relatively high concentration of $O_2$ in chloroplasts. To cope with oxidative stress, plants have evolved an efficient ROS-scavenging enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), and low molecular weight antioxidants including ascorbate, glutathione and phenolic compounds. To maintain the productivity of plants under the stress condition, it is possible to fortify the antioxidative mechanisms in the chloroplasts by manipulating the antioxidation genes. A powerful gene expression system with an appropriate promoter is key requisite for excellent stress-tolerant plants. We developed a strong oxidative stress-inducible peroxidase (SWPA2) promoter from cultured cells of sweetpotato (Ipomoea batatas) as an industrial platform technology to develop transgenic plants with enhanced tolerance to environmental stress. Recently, in order to develop transgenic sweetpotato (tv. Yulmi) and potato (Solanum tuberosum L. cv. Atlantic and Superior) plants with enhanced tolerance to multiple stress, the genes of both CuZnSOD and APX were expressed in chloroplasts under the control of an SWPA2 promoter (referred to SSA plants). As expected, SSA sweetpotato and potato plants showed enhanced tolerance to methyl viologen-mediated oxidative stress. In addition, SSA plants showed enhanced tolerance to multiple stresses such as temperature stress, drought and sulphur dioxide. Our results strongly suggested that the rational manipulation of antioxidative mechanism in chloroplasts will be applicable to the development of all plant species with enhanced tolerance to multiple environmental stresses to contribute in solving the global food and environmental problems in the 21st century.

물오리나무의 뿌리혹으로부터 Frankia 홍생규주의 (洪生菌株) 분리

권석윤(Seok Yoon Kwon),안정선(Chung Sun An) 한국식물학회 1989 Journal of Plant Biology Vol.32 No.1

An endophyte was isolated from the root nodule of Alnus hirsuta. The isolated endophyte was identified as a Frankia sp. through morphological characteristics. Their infectivity and effectivity were confirmed by introgen-fixing root nodules induced on inoculated Alnus seedlings. Reisolated endophyte from the induced nodule showed identical morphological characteristics as the first isolate, showing the nodule was induced by the first isolate. Consequently, the first isolate was confirmed as a true symbiont of Alnus hirsuta root nodule. The isolate was designated as a Frankia SNU 014201 strain.

양배추 RNA-Seq 데이터를 이용한 full length transcript assembly

김상미,최준경,이봉우,권석윤,김혜란,조성환 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

고품질의 표준 유전자 세트를 확보하는 것은 작물의 기능연구 수행에 매우 중요하다. 고품질의 표준 유전자 세트란 형성된 transcripts의 assembly의 정확성이 높고, full length transcript의 비율이 높은 유전자 세트를 뜻한다. 본 연구에서는 표준 유전체가 보고되지 않은 양배추의 고품질 표준 유전자 세트 확보를 목표로 하며, 그 중 full length transcript 비율이 어느 정도 인지를 측정하고자 한다. 본 연구에서 full length transcript는 표준 유전체에 90% 이상 일치하며, 전사체의 5’UTR과 3’UTR 모두 가진 transcript로 정의하고 분석을 수행하였다. 우선 표준 유전체가 있는 애기장대를 이용하여 full length transcript의 비율 측정하였다. de novo 어셈블리로 애기장대의 표준 유전자 세트를 작성한 결과 26,255 transcripts가 형성되었으며, 26,084 transcripts가 표준 유전체에 90% 이상 일치됨을 확인했다. 그 중 91%에 해당하는 transcripts는 알려진 전사체 13,852개와 매칭이 되었는데, 43%에 해당하는 10,231 transcripts가 전사체 13,852개의 full length transcript로 확인되었다. 위와 동일한 방법을 적용하여 양배추의 표준 유전자 세트를 형성한 결과 53,562 transcripts를 형성되었다. 양배추는 표준 유전체의 부재로 알려진 식물 30종의 아미노산 서열을 이용하여 분석하였다. 그 결과 35,273 transcripts가 알려진 전사체와 매칭이 되었으며, 그 중 45%에 해당하는 15,765 transcripts가 full length transcripts임을 확인하였다. 이 비율은 이미 보고된 유칼립투스와 제브라피시의 transcriptome assembly를 통해 얻어진 full length transcripts 6,208(39.5%), 9,625(26%) 보다 더 향상된 수준이다. 본 연구에서 수행된 방법론을 다른 작물의 표준 유전자 세트 형성에 적용하였을 때 보다 향상된 고품질 표준 유전자 세트를 생산할 것으로 예측된다.

Superoxide Dismutase 와 Ascorbate Peroxidase 를 엽록체에 과발현하는 형질전환 담배의 수분스트레스에 대한 반응

최선미,권석윤,곽상수,박용목 한국환경과학회 2001 한국환경과학회지 Vol.10 No.1

To assess resistance of transgenic tobacco plants which overexpress superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts to water stress, changes in leaf water potential, turgor potential, stomatal conductance and transpiration rate were measured. Leaf water potential in all plants remained high up to day 4 after withholding water but thereafter decreased markedly. In spite of a remarkable decrease in leaf water potential, some of transgenic plants maintained higher turgor potential compared with control plant on day 12. In particular, the transgenic plant expressing MnSOD showed an outstanding maintenance in turgor pressure by osmotic adjustment throughout the experiment, resulting in high stomatal conductance and transpiration rate. However, among transgenic plants, osmotic potential was reduced more effectively in multiple transformants such as the double transformant expressing both MnSOD and APX, and the triple transformant expressing CuznSOD, MnSOD and APX than single transformants. Consequently, further research is needed to get general agreement on the tolerance of transgenic plants to water stress at different growth stages for each transgenic plant.

SOD와 APX를 동시에 엽록체에 발현시킨 형질전환 감자 (cv. Superior)의 산화스트레스 내성 증가

탕리,권석윤,김명덕,김진석,곽상수,이행순,Tang, Li,Kwon, Suk-Yoon,Kim, Myoung-Duck,Kim, Jin-Seog,Kwak, Sang-Soo,Lee, Haeng-Soon 한국식물생명공학회 2007 식물생명공학회지 Vol.34 No.4

산화스트레스 유도성 SWPA2 프로모터 조절하에 항산화 효소 SOD와 APX 유전자를 동시에 엽록체에 발현시킨 형질전환 감자 (품종 수미)를 대상으로 methyl viologen (MV) 처리에 의해 유도되는 산화스트레스 내성을 잎절편체, 소식물체 및 식물체 수준에서 조사하였다. 잎 절편에 $3{\mu}M$ MV를 처리하였을 때 SSA 식물체의 잎절편체는 비형질전환 (NT) 식물체에 비해 40% 정도 상해를 적게 받았다. 소식물체 수준에서 MV에 의한 산화스트레스 내성을 조사하기 위하여 SSA감자 shoot을 $0.3{\mu}M$ MV 첨가 배지에 배양하였을 때 뿌리의 생장에서 내성이 나타났다. 또한 온실에서 4주 생장한 식물체에 $350{\mu}M$ MV를 처리하였을 경우에도 SSA 식물체는 NT 식물체에 비해 약 75% 손상을 적게 입은 것으로 나타내었다. 추후 SSA 식물체를 이용하여 건조, 고온 등의 복합재해에 내성을 분석이 진행되어야 할 것이며 그 결과 복합스트레스 내성 감자 품종 (수미)을 개발할 수 있을 것으로 기대한다. Oxidative stress is a major damaging factor for plants exposed to environmental stresses. Previously, we have generated transgenic potato (cv. Superior) plants expressing both CuZnSOD and APX genes in chloroplast under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants) and selected the transgenic potato plant lines with tolerance against methyl viologen (MV)-mediated oxidative stress. When leaf discs of SSA plants were subjected to $3{\mu}M$ methyl viologen (MV), they showed approximately 40% less damage than non-transgenic (NT) plants. SSA plantlets were treated with $0.3{\mu}M$ MV stress, SSA plants also exhibited reduced damage in root growth. When 350 MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage, which was approximately 75% less damage than leaves of NT plants. These plants will be used for further analysis of tolerance to environmental stresses, such as high temperature and salt stress. These results suggest that transgenic potato (cv. Superior) plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions.

High-yield Production of Functional Human Lactoferrin in Transgenic Cell Cultures of Siberian Ginseng (Acanthopanax senticosus)

이행순,권석윤,박두상,양경실,김재훈,이기택,곽상수,Seung-Hyun Jo 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.5

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

체세포배발생을 통한 국내 주요 고구마 품종의 식물체 재분화

권은정,권석윤,김문자,안영섭,이준설,정병춘,곽상수,이행순,Kwon, Eun-Jeong,Kwon, Suk-Yoon,Kim, Moon-Za,Lee, Joon-Seol,Ahn, Young-Sup,Jeong, Byeong-Choon,Kwak, Sang-Soo,Lee, Haeng-Soon 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.3

4종류 고구마 품종을 대상으로 정단분열조직 배양에 의한 배발생 캘러스 유도, 이들로부터 고빈도의 체세포배발생과 식물체 재분화 체계를 확립하였다. 자미. 율미, 신황미, 및 화이트 스타의 생장점을 LS 기본배지에 1 mg/L auxin (2,4-D, picloram, dicamba)가 첨가된 배지에서 배양하였다. 그 결과 2,4-D 첨가 배지에서 가장 높은 빈도로 배발생 캘러스가 유도되었으며, 배양 4주째에 유도된 배발생 캘러스의 빈도는 신황미, 자미, 율미. 및 화이트 스타에서 86%, 78%, 76%, 및 80%를 각각 나타내었다. 2,4-D 첨가 배지에서 얻어진 배발생 캘러스를 2.4-D를 제거한 배지로 옮겨 주어 체세포배를 유도하였으며, 이 체세포배의 90% 이상이 완전한 식물체로 발달하였다. 이러한 결과는 정단분열조직 배양으로부터의 체세포배 발생 방법이 광범위한 고구마 품종에 적용될 수 있을 뿐만 아니라 분자육종을 통한 신기능성 고구마 품종개발에 효율적으로 이용될 수 있을 것으로 기대된다. An efficient plant regeneration system of major cultivars of sweetpotato (Ipomoea batatas (L.) Lam.) in Korea via somatic embryogenesis was established. Embryogenic calli were formed from shoot apical meristems of sweetpotato cultivars when cultured on LS medium supplemented with 1 mg/L auxin (2,4-D, picloram, dicamba). Among three kinds of auxin, 1 mg/L 2,4-D showed the highest embryogenic calli induction rate. After 4 weeks of cultures on LS medium supplemented with 1 mg/L 2,4-D, embryogenic calli induction rates of Sinhwangmi, Zami, Yulmi, and White Star were 86%, 78%, 76%, and 80%, respectively. Upon transfer onto LS basal medium, most of somatic embryos developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to mature plants in a greenhouse.

CuZnSOD와 APX를 엽록체에 발현시킨 담배식물체의 Highlight와 Chilling 스트레스에 대한 광합성 효율

김윤희,권석윤,곽상수,방재욱 한국식물생명공학회 2003 JOURNAL OF PLANT BIOTECHNOLOGY Vol.30 No.4