콩으로부터 상처 유도 beta-amyrin synthase 유전자의 동정 및 발현분석

박성환,이재헌,Park, Seong-Whan,Lee, Jai-Heon 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.2

Suppression subtractive hybridization (SSH)를 통해 상처에 의해 발현이 유도되는 cDNA들을 분리하였고, 그 중 하나인 gmwi33은 $\beta$-amyrin synthase 유전자들과 높은 유사성을 보였다. gmwi33의 전장 cDNA인 GmAMS1은 2416 bp 길이에 739개 아미노산으로 구성된 긴 open reading frame(ORF)를 포함하고 있었다. GmAMS1 단백질은 감초의 $\beta$-amyrin synthase인 GgbAS와 89%, 완두의 OSCPSY와 86%의 유사성을 보였다. 암조건 하에 5일간 기른 콩나물에서, GmAMS1는 빛을 쪼여주었을 때 가장 강하게 발현되었고 methyl jasmonate 처리와 저온처리 시에도 발현이 유도된 반면, UV-B나 elicitor를 처리하였을 때는 발현이 유도되지 않았다. 이러한 GmAMS1의 발현양상은 사포닌의 활성산소 제거기능과 밀접한 연관이 있을 것으로 추측된다. Suppression subtractive hybridization (SSH) was used to isolate wound-induced cDNAs from wounded soybean. One of wound-induced cDNA, gmwi33 showed high homology with genes encoding $\beta$-amyrin synthase. The full length cDNA of gmwi33, designated GmAMS1, is 2416 bp long and contains an open reading frame consisted of 739 amino acids. GmAMS1 protein showed 89% identity with licorice GgbAS1 and 86% identity with pea OSCPSY. In 5 day-old, dark-grown seedlings, the expression of GmAMS1 was most strongly induced by light and weakly induced by methyl jasmonate and by low temperature. However, GmAMS1 was not induced by elicitor or UV-B treatment. Such expression pattern might be closely related with the oxygen-radical scavenging activity of soyasaponin.

저온 스트레스에 발현이 유도되는 콩의 L-asparaginase 유전자의 분리

박성환,김기영,진량,이재헌,Park, Seong-Whan,Kim, Kee-Young,Chen, Liang,Lee, Jai-Heon 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.2

Suppression subtractive hybridization (SSH)를 통해 저온에 의해 발현이 유도되는 cDNA들을 분리하였고, 그 중 하나인 slti182는 asparaginase 유전자들과 높은 유사성을 보였다. Slti182의 전체 cDNA인 GmASP1은 1258 bp 길이에 326개 아미노산으로 구성된 긴 Open reading frame (ORF)를 포함하고 있었다. GmASP1 단백질은 애기장대의 putative asparaginase (AB012247)와 84%의 유사성을 보였으나, 애기장대의 다른 isoform(Z34884)와는 55%의 유사성을 나타내었다. GmASP1 유전자는 저온 처리 후 3시간째부터 발현이 유도되어 6시간째에 최대발현을 나타내었고, 이후 점차 감소하여 48시간에는 저온처리전 수준으로 되돌아왔다. 또한, 식물체를 저온에서 48시간 둔 후에 다시 상온으로 옮겼을 때, 정상수준으로 떨어진 GmASP1의 발현이 다시 증가하는 양상을 보였다. 이러한 결과로 볼 때, GmASP1이 저온 스트레스에 반응 초기의 단백질 합성 촉진에 중요한 역할을 할 것으로 사료된다. Suppression subtractive hybridization (SSH) was used to isolate wound-induced cDNAs from wounded soybean. One of low-temperature-inducible cDNA, slti182 showed high homology with genes encoding 1-asparaginase. The full length cDNA of slti182, deginated GmASP1, is 1258 bp long and contains an open reading frame consisted of 326 amino acids. CmASP1 protein showed the highest identity (84%) with putative asparaginase from A. thaliana (AB012247), but it showed only 55% identity with another isoform of A. tathaliana (Z34884). The expression of GmASP1 during low temperature stress started to increase 3 hours after treatment, reached the maximum at 6 hour, and then decreased to the initial level at 48 hours. The amount of GmASP1 transcripts increased again when low-temperature-treated plants were transferred to room temperature, The present study suggests that GmASP1 may function to accelerate the protein synthesis which is important in the early response to low temperature.

Teratogenic Inducing Capacity of Aloe Extract on Different Stages of Zebrafish Embryos

Manigandan Krishnan(마니간단 크리쉬난),Jai Heon Lee(이재헌),Sun Chul Kang(강선철) 한국약용작물학회 2016 한국약용작물학회 학술대회논문집 Vol.2016 No.2

밀과 보리 原形質體의 融合 硏究

Heung Bae Kim(金興培),Jai Heon Lee(李在憲) 한국육종학회 1987 한국육종학회지 Vol.19 No.1

Factors affecting isolation and fusion of mesophyll tissue from T. aestivum and H. bulbosum for producing somatic cell hybrids were studied. In case of both wheat and barley, the highest protoplast yield was obtained when tissues were submitted to Mannitol 0.7 M for 2 hours. High protoplast yield was resulted when a solution of Cellulase 3%, Macerozyme 0.8% and Pectolyase 0.03% was used for 4 hours. The favorable pH of Mannitol and enzyme solution was 5.8. The most effective concentration of PEG ranged from 30% to 40% for fusion of T. aestivum and H. bulbosum mesophyll protoplasts. The best density of protoplast was 2.5×10⁵/ml for cell fusion.

국내 거베라 육종계통 형질전환 기초 조건 확립

이혜영,이기정,전은희,신상현,이재헌,김도훈,정대수,정용모,조용조,김정국,정영수,Lee, Hye-Young,Lee, Ki-Jung,Jeon, Eun-Hee,Shin, Sang-Hyun,Lee, Jai-Heon,Kim, Doh-Hoon,Chung, Dae-Soo,Chung, Yong-Mo,Cho, Yong-Cho,Kim, Jeong-Kook,Chung, You 한국식물생명공학회 2006 식물생명공학회지 Vol.33 No.1

본 연구에서는 거베라 형질전환 기초기술개발을 위한 연구를 수행하였다. 경남원예화훼시험장에서 육성된 국내 거베라 18품종 중, Agrobacferium 감염에 순응적인 품종을 선발하기 위하여 18개의 국내 거베라 품종을 스크린하여 011, 016, GM023 등 12개의 순응형 계통을 스크린 하였으며 효율적인 형질전환을 위한 제균 항생제로는 cefotaxime이 선택되었다. 더 많은 캘러스 유도를 위한 전처리 배지의 호르몬 농도는 BA 2 ppm, Zeatin 2 ppm, IAA 0.2 ppm (2 x media)이 효율적이었고 재분화 배지의 호르몬 농도도 전처리 배지 호르몬의 농도와 동일하게 BA 2 ppm, Zeatin 2 ppm, IAA 0.2 ppm (2 x media)에서 효율적이었으나 GUS 발현에는 다른 차이가 없었다. 고효율 형질전환 체계 확립을 위하여 네 가지 다른 전처리 기간 처리와 두 가지 다른 Agrobacterium tumerfaciens 사용, 두 가지 다른 공배양 기간을 처리한 결과 전처리 하지 않은 엽병과 엽신을 LBA4404 Agrobacterium tumerfaciens을 사용하여 5일 간 공배양 기간을 가졌을 때와 dipping 처리하여 접종한 엽병과 엽신에서 높은 GUS 발현빈도를 나타냈다. Gerber (Gerbera hybrida) is a valuable ornamental species grown as a potted plant and cut flowers. However, genetic variability within the gerbera genus is very limited. So it is absolutely needed to introduce and widen genetic resources into gerbera lines by genetic transformation. For the purpose, 18 Korean gerbera lines were screened to establish Agrobacterium-mediated genetic transformation procedure. In an experiment to select Korean gerbera lines which are amenable to Agrobacterium-inoculation, 12 lines turned out to be positive in Agrobacterium-inoculation. More callus were produced from BA 2ppm, Zeatin 2ppm, IAA 0.2ppm in pre-culture and regeneration medium (2X media) but there was no difference in the frequency of GUS expression rate. In another experiment to find out optimal condition for highly efficient Agrobacterium-inoculation, petiole and leaf explants have been treated with four different pre-culture periods, two different co-culture periods and two different Agrobacterium strains. As a result, high GUS expression has been showed from petiole and leaf explants treated no pre-culture period with LBA4404 Agrobacterium tumerfaciens, 5 day co-culture period and dipping treatment.

RAPD를 이용한 들깨 유전자원의 유전적 변이 분석

김도훈,양보경,김현경,김나영,정순재,김익수,남재성,이재헌,정대수,Kim, Doh-Hoon,Yang, Bo-Kyung,Kim, Hyeon-Kyoung,Kim, Na-Young,Jeong, Soon-Jae,Kim, Ik-Soo,Nam, Jae-Sung,Lee, Jai-Heon,Chung, Dae-Soo 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.3

Genetic variation of Perilla germplasms was investigated using RAPD markers. Forty-two Perilla frutescens lines and cultivars collected form locals were subjected to RAPD analysis using 220 primers. Among them only 13 primers showed polymorphic bands and these 13 primers provided a total of 144 bands, consist of 115 polymorphic and 29 monomorphic ones. The polymorphic bands were subjected to phylogenetic analysis using UPGMA and maximum parsimony (MP) methods. In the UPGMA method, similarity coefficiency of 42 Perilla frutescens lines and cultivars ranged from 0 to 0.7842. The dendrogram of 42 lines and cultivars obtained through UPGMA method resulted in two major groups, and the similar clustering pattern was found by MP method, suggesting Perilla germplasms utilized in this study truly can be divided into two major groups. Although the two major groups were consistent roughly with their phenotypes (under of node, weight of 1,000 grains, and oil content), in detail, much inconsistency also was present.

국내 콩(Glycine max) 품종 형질전환 초기조건 확립

이기정,서진경,이혜영,전은희,신상현,이재헌,김도훈,고종민,한원영,백인열,오병준,정영수,Lee Ki-Jung,Seo Jen-Kyung,Lee Hye-Young,Jeon Eun-Hee,Shin Sang-Hyun,Lee Jai-Heon,Kim Doh-Hoon,Ko Jong-Min,Hahn Won Young,Baek In-Youl,Oh Boung-Jun,Chung 한국생명과학회 2006 생명과학회지 Vol.16 No.2

경제적으로 중요한 식용작물인 콩(Glycine max)의 초기형 질전환 효율증대를 위하여 연구를 수행하였다. 높은 초기형 질전환 조건 확립을 위하여 기초실험으로 Agrobacterium 감 염에 순응적인 국내 콩 품종을 스크린 하였으며, 적정한 agar 농도와 선발항생제 hygromycin 농도 규명, 상처방법, explant의 치상방향, 호르몬 전처리, 액체배지 선발 등 형질 전환 초기효율에 미치는 영향을 transient GUS 분석을 통하여 알아보았다. Agrobacterium 감염에 순응적인 품종을 선발하기 위하여 32개의 국내 콩 장려품종을 스크린한 결과, 일품검정콩, 은하콩, 만리콩, 대원콩 등 14개의 순응형 품종을 선발하였으며, 효율적인 제균제로는 cefotaxime이, 효율적인 선발항생제로서는 hygromycin이 선택되었다. Hygromycin 농도는 10-15 ppm이 효율적이었고, agar 농도 0.6-0.8%, explant의 치상방향은 향배축(adaxial side)을 down하여 치상하였을 때가 높은 GUS ($\beta$-glucuronidase)발현빈도를 나타냈고 상처방법은 scalpel보다. 동양침을 사용하였을 때 높게 나타났다. 형질전환 체계확립을 위하여 호르몬 전처리를 한 결과, BA 5 ppm 과 10 ppm을 처리하였을 때 후기 선발에서 높은 생존율을 보였고, 전처리 후에 액체배지에서 선발을 하였을 때 비형질 전환체(escape)의 빈도를 크게 낮출 수 있었다. 또한 여러 조건의 결과를 비교하여 볼 때, 국내 콩 품종 중에서 형질전환을 위한 재료로써는 은하콩이 가장 적합한 것으로 생각된다. In order to establish highly efficient gene transfer condition at early stage of soybean transformation, various experiments were performed and compared their efficiencies by transient GUS analysis; those conditions are genotype determination of Korean soybean cultivars for amenability to Agro-infection, appropriate agar and selective agent concentration, orientation of explant placement, hormone pre-culture, and liquid selection condition. In the genotype screen of Korean soybean varieties, 14 amenable genotypes were selected. For efficient Agrobacterium washing, cefotaxime was chosen and hygromycin at the concentration of 10 and 15 ppm was used as selection agent in the media. Agar concentration was slightly better in 0.6% and 0.8% for both shoot and callus formation, and explant placement with adaxial side down showed high frequency of GUS expression. For wounding treatment, oriental needle was efficient than scalpel for shoot formation and gene transfer. To increase the frequency of gene transfer, hormone pre-treatment was applied. BA at the concentration of 5 and 10 ppm resulted in better survival at the late stage of selection in shoot elongation media. Selection in liquid media after hormone pre-treatment seemed to be effective to remove the escaped non-transformants at early stage of procedure. Considering the results obtained, Eunhakong could be the first choice as a material for soybean transformation among Korean soybean genotypes.

백선의 형태적 특성, 항균 효과 및 분자 세포학적 분석

정대수,조창우,김경미,허지은,김수영,정영수,김도훈,방재욱,최정,이재헌,Chung, Dae-Soo,Cho, Chang-Woo,Kim, Kyung-Mee,Heo, Jee-Eun,Kim, Soo-Young,Chung, Young-Soo,Kim, Doh-Hoon,Bang, Jae-Wook,Cui, Zheng,Lee, Jai-Heon 한국생명과학회 2006 생명과학회지 Vol.16 No.7

The importance of wild plant resources along with the development of high biotecyhnology is highlighted for exploitation of new materials which can make the added value. The goal of this study is providing fundamental data bases for developing new materials through the analyses of morphological characteristics, antibiotics and molecular cytogenetics in Baegseon (Dictamnus dasycarpus Turcz.). Baegseon has several characteristics; there are two types of flower color, pink and white, the seed germination starts about February 20th, the maximum flowering season is around May 17th in southern Korea, the growth period is about 60 days. The number of chromosomes are 2n=2x=36, the size of chromosomes in metaphase is $4.2{\sim}8.1{\mu}m$. The amount of 2C nuclear DNA is 1.93pg, and there is no variation of genome size amoung varieties. The extraction juice of baegseon young roots has the excellent antibiotic activity against the mold (Botrytis cinerea).

유자와 탱자 과피 추출물의 항산화 및 면역 활성 효과

박준희(Joon Hee Park),강병원(Byoung Won Kang),김지은(Ji Eun Kim),서민정(Min Jeong Seo),이영춘(Young Choon Lee),이재헌(Jai Heon Lee),주우홍(Woo Hong Joo),최영현(Yung Hyun Choi),임학섭(Hak Seob Lim),정영기(Yong Kee Jeong),이복규(Bok Ky 한국생명과학회 2008 생명과학회지 Vol.18 No.3

본 연구는 탱자(Poncirus trifoliata) 과피의 80% 에탄올 추출물(PTP)에 대한 항산화 활성과 면역 활성을 유자(Citrus junos) 과피 추출물(CJP)과 비교하였다. 총 phenol 함량과 총 flavonoids 함량은 PTP가 각각 60.75±1.15 ㎎/100 g과 33.75±0.15 ㎎/100 g이었으며, 이는 CJP에 비해 다소 낮았다. PTP의 항산화 활성은 농도 의존적으로 증가하였으며 CJP의 항산화 활성과 비슷하게 나타났다. 면역반응 중 대식세포가 생성하는 NO 생성량을 측정한 결과 CJP와 PTP를 단독으로 처리한 경우와 LPS와 혼합하여 처리한 경우 모두 1㎎/ml까지는 대조구와 비교하여 농도 의존적으로 증가하였으나 고농도에서는 감소하는 경향을 나타내었다. 비장세포를 이용한 면역세포 증식능에서 CJP와 PTP를 첨가한 경우, 1 ㎎/ml까지는 대조구와 비교하여 농도 의존적으로 증가하였으나 고농도에서는 감소하였다. In this study, we compared with 80% ethanol extracts from peel of Poncirus trifoliata (PTP) and peel of Citrus junos (CJP) against antioxidant and immune activities. Total phenolics and flavonoids contents in PTP extracts were 60.75±1.15 and 33.75±0.15 ㎎/100 g, respectively, and those were lower than CJP extracts. Antioxidant activities of PTP were increased with the more concentration, and were similar to CJP. Antioxidant activities of PTP were increased with increasing of concentration, and were similar to those of CJP. The NO production in macrophage cell lines were increased in a dose-dependent manner, until 5 ㎎/ml of CJP and 1 ㎎/ml of PTP compared with control cells, but decreased at higher concentrations. The proliferation of mouse spleen cells were increased in a dose-dependent manner, until 1 ㎎/ml of CJP and PTP compared with control cells but decreased at higher concentrations. The NO production in macrophage cell lines treated with PTP and CJP were increased in a dose-dependent manner, compared with untreated control cells until the concentrations of 1~5 ㎎/ml (CJP) and 1 ㎎/ml (PTP) but decreased at higher concentrations than that. The proliferation of mouse spleen cells treated with PTP and CJP were increased in a dose-dependent manner, compared with untreated control cells until the concentration of 1 mg/ml but decreased at higher concentrations than that.

β-lactam계 항생물질 저항성을 지닌 Bacillus sp. J105 균주로부터 분비되는 베타 락탐 분해효소의 정제 및 특성

조경순(Kyeong Soon Cho),강병원(Byoung Won Kang),서민정(Min Jeong Seo),이영춘(Young Choon Lee),이재헌(Jai Heon Lee),주우홍(Woo Hong Joo),최영현(Yung Hyun Choi),임학섭(Hak Seob Lim),김정인(Jeong In Kim),서권일(Kwon Il Seo),정영기(Yong 한국생명과학회 2008 생명과학회지 Vol.18 No.6

Bacillus sp. J105 strain으로부터 유도된 β-lactamase는 ammonium sulfate 침전, 이온 교환 칼럼 크로마토그래피, 겔 여과 등의 과정을 거쳐 SDS-PAGE에서 단일 band로 정제하였다. 정제된 효소의 분자량은 31 kDa이었으며 등전점은 7.35이었다. 효소반응의 최적 pH와 온도는 각각 5와 40℃이었다. 정제 단백질의 총 아미노산 조성의 분석 결과, Gly과 Ala이 각각 14.1과 13.3 mole%로 가장 많은 아미노산 잔기를 차지하고 있었다. 정제 효소의 ampicillin을 기질로 하였을 때의 Km값은 1.33 mM이었고 Vmax값은 0.36 mM/㎖이었다. β-Lactamase, secreted from Bacillus sp. J105 strain was purified to a single band on SDS-PAGE by ammonium sulfate precipitation, ion exchange column chromatography and gel-filtration. The molecular weight of the purified enzyme was 31 kDa on SDS-PAGE and its isoelectric point was 7.35. Optimal pH and temperature for enzymatic reaction were 5 and 40℃, respectively. As a result of total amino acid composition analysis of the purified enzyme, Gly and Ala were occupied 14.1 and 13.3 mole %, respectively. Km and Vmax value of purified enzyme were 1.33 mM and 0.36 mM/㎖ using ampicillin as a substrate, respectively.