효모 발현 시스템에서의 고구마 peroxidase (SWPA2) 프로모터 활성분석

허경혜 仁濟大學校 2005 仁濟論叢 Vol.20 No.1

peroxidase (POD, E.C.1.11.1.7)는 식물을 포함한 생물계 전체에 존재하며, POD는 일반적으로 외부의 다양한 조건에 의해 그 활성이 증가되기 때문에 환경스트레스의 내성인자로 간주된다. 고구마 현탁 배양세포에서 강하게 발현되는 SWPA2 유전자의 프로모터 특성을 규명한 결과, SWPA2 프로모터는 산화스트레스에 의해 발현이 조절되는 산업적 가치가 높은 promoter임이 확인되었다. 본 연구에서는 식물 현탁 배양세포에서 강하게 발현하는 SWPA2 프로모터의 활성을 분자생물학의 모델시스템인 효모에서 살펴보았다. SWPA2 프로모터는 효모에서 강한 활성을 보였으며 산화스트레스원에 반응하여 프로모터 활성이 증가하였다. 그러나 SWPA2 프로모터를 가지는 형질전환식물체와 비교하여 볼 때 효모에서 산화스트레스 하에서 SWPA2 프로모터 활성 유도는 미비하였다. 결론적으로 고구마 POD프로모터를 효모에서 산업적으로 이용할 수는 있으나 산화스트레스에 반응하는 조절인자를 밝히는 시스템으로는 적합하지 않음을 알 수 있었다.

한국 달맞이꽃의 Isozyme Analysis 및 Fatty Acid Composition에 관한 연구

허홍욱,허경혜 釜山大學校 師範大學 1993 교사교육연구 Vol.27 No.-

The genetic variation in the Korean populations of Everning Primrose, Oenothera odorata L., was examined to check out the frequency of allozyme variation. The allozyme variation of ADH, MDH, 6-PGDH, PGM, and IDH were assayed by means of starh gel electrophoresis. The ADH, 6-PGDH and PGM loci were almost monomorphic which was under control of one locus. Only Seol-ag population shows slow allele(3.0%) at the ADH-locus. At the MDH-2 locus, the frequency of slow and fast allele were 5.9% and 4.5% on the average, this represented a single locus with two alleles for a dimeric enzyme. And also IDH-locus were 0.7% and 3.6% respectively. Fatty acid comosition were investigated by means of capillary column gas-chromatography. The fatty acid of Everning Primrose oil was composed of palmitic(5.79-7.48), stearic (1.51-1.79%), oleic(4.15-7.81%), and linoleic(71.37-76.38%) acid. Particularly, GLA which is a precursor of prostaglandin was 10.15-12.51% among total fatty acid.

토마토(Lycopersicon esculentum Mill) 현탁배양세포에서 Superoxide Dismutase 활성

유순희,허경혜,권석윤,이행순,방재욱,곽상수 한국식물생명공학회 1997 식물생명공학회지 Vol.24 No.1

Superoxide dismutase (SOD) 고생산세포주로 선발된 토마토(Lycopersicun esculentum) 배양세포를 사용하여 현탁배양에 따른 SOD 활성과 isoenzyme변화를 조사하고 토마토 식물체의 것과 비교하였다. 현탁배양은 세포생중량 2 g을 1 mg/L 2,4-D, 30 g/L sucrose를 함유한 MS 배지 50 mL과 함께 mL flask에서 $25^{\circ}C$암상태로 배양(100 rpm)하였다. 세포생장은 계대배양후 20일에 최고점에 도달한 후, 급격히 감소하며 배양 후 23일부터 세포가 검게 변하였다. 세포 단위무게당 SOD활성(unit/g dry cell wt)은 배양 후 23일부터 증가하여 28일째에 최고활성(52,400 unit)을 나타낸 후 급격히 감소하였다. 세포 밖으로 분비되는 extracellular SOD활성은 배양 후 25일에 최고치(27,800 unit/so mL medium)를 나타낸 후 감소하였다. Flask 전체의 SOD활성은 배양 후 25일에 최대치(35,700 unit)를 나타내었으며 extracellular SOD 활성이 약 75%을 차지하였다. 토마토 배양세포에는 4개의 SOD isoenzyme이 존재하며, isoenzyme의 패턴변화는 세포생장에 따른 효소활성의 변화와 일치하였다. 토마토 식물체는 배양세포에 없는 CuZnSOD가 존재하며 배양세포와 식물체 조직사이에는 서로 다른 isoenzyme 패턴이 존재함을 알 수 있었다. We investigated changes in the superoxide dismutase (SOD) activity and SOD isoenzyme pattern in suspension cultures of tomato (Lycopersicon esculentum), which were compared with those of intact tomato plants. two grams (fr wt) of cells subcultured at 15-day intervals were inoculated into 50 mL MS medium containing l mg/L 2,4-D and 30 g/L sucrose in a 300 mL flask and maintained at $25^{\circ}C$ in the dark (100 rpm). The cell growth reached a maximum at 20 days after subculture (DAS), followed by a rapid decrease with further cultures. The cell colour changed from white to black from 23 DAS. The intracellular SOD activity (units/g cell dry wt) was significantly increased from 23 DAS and reached a maximum at 28 DAS (52,400 units), followed by a decrease with further cultures, whereas the extracellular SOD activity showed a maximum at 25 DAS (27,800 units/50 mL medium). The total SOD activity per flask showed a maximum at 25 DAS (35,700 units), in which the extracellular SOD activity occupied about 75%. The tomato cultured cells had four SOD isoenzymes and their patterns were well correlated with SOD activity without a qualitative change during the cell cultures. The intact tomato plants had an additional CuZnSOD isoenzyme, showing the different isoenzyme patterns from cultured cells.

Effects of Clonidine on the Negative Chronotropic Response Induced by Vagal Stimulation in the Rat

홍승철,허경혜,정준기,박미선 朝鮮大學校 1988 藥學硏究誌 Vol.22 No.2

Members of the ran family of stress-inducible small GTP-binding proteins are differentially regulated in sweetpotato plants

김영화,허경혜 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.1

Ran is a small GTP-binding protein that binds and subsequently hydrolyzes GTP. The functions of Ran in nuclear transport and mitotic progression are well conserved in plants and animals. In animal cells, stress treatments cause Ran relocalization and slowing of nuclear transport, but the role of Ran proteins in plant cells exposed to stress is still unclear. We have therefore compared Ran genes from three EST libraries construed from different cell types of sweetpotato and the distribution pattern of Ran ESTs differed according to cell type. We further characterized two IbRan genes. IbRan1 is a specific EST to the suspension cells and leaf libraries, and IbRan2 is specific EST to the root library. IbRan1 showed 94.6 % identity with IbRan2 at the amino acid level, but the C-terminal region of IbRan1 differed from that of IbRan2. These two genes showed tissue-specific differential regulation in wounded tissues. Chilling stress induced a similar expression pattern in both IbRan genes in the leaves and petioles, but they were differently regulated in the roots. Hydrogen peroxide treatment highly stimulated IbRan2 mRNA expression in the leaves and petioles, but had no significant effect on IbRan1 gene expression. These results showed that the transcription of these two IbRan genes responds differentially to abiotic stresses and that they are subjected to tissue-specific regulation. Plant Ran-type small G-proteins are a multigenic family, and the characterization of each Ran genes under various environmental stresses will contribute toward our understanding of the distinctive function of each plant Ran isoform.

Molecular Characterization and Expression of the Cinnamyl Alcohol Dehydrogenase Gene Family in Sweet Potato (Ipomoea batatas) under Environmental Stresses

김영화,허경혜 한국원예학회 2019 원예과학기술지 Vol.37 No.2

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of monolignol biosynthesis and isa key enzyme for lignification. Most of the CAD genes in angiosperms belong to a multigenefamily. As in other plants, 13 cDNAs encoding CAD (IbCADs) were isolated from the expressedsequence tag (EST) library of sweet potato (Ipomoea batatas). A phylogenetic analysis revealedthat IbCADs belonged to the nondevelopmental CAD proteins, which are not conserved as bonafide CADs. The IbCAD proteins were classified into four groups (Groups I-IV) according to theiramino acid similarity. In all groups, IbCADs contain the Zn-binding domains, suggesting that theybelong to a family of zinc-dependent alcohol dehydrogenases. Each group showed sequencedivergence in the residues related in substrate specificity. Different structural characteristics ofIbCADs were supported by their different transcriptional expression patterns. The IbCAD genes infour groups were highly induced in response to environmental stresses (cold, H2O2, and wounding),but each group showed different patterns and levels of expression. The differential response of theIbCAD gene family under different stresses might indicate a more complex nature of the CAD geneexpression in sweet potato. These diverse expression patterns represented an important function ofeach IbCAD gene in tolerance to environmental stresses. To the best of our knowledge, this is thefirst study to characterize genes of the CAD family genes in a root crop. These results could beuseful in understanding the physiological roles and characteristics of the IbCAD multigene family.

Acetylcholinesterase 효소 저해도와 GC/MS 방법을 이용한 녹차의 잔류농약 분석

김영화,허경혜 한국차학회 2013 한국차학회지 Vol.19 No.2

본 연구에서는 AChE의 저해도와 GC/MS 분석방법을 이용하여 녹차의 잔류농약(chlorpyrifos, fenitrothion, methidathion)을 분석하였다. Chlorpyrifos의 최대잔류허용량 (MRL)2 ppm에서 AChE의 활성이 75% 저해되었고 여기에 녹차추출물이 첨가된 경우에는 83% 저해를 나타내었다. 그러나 AChE 활성저해는 2 ppm보다 훨씬 낮은 농도에서 저해도 값에 변화를 보이지 않았다. Chlorpyrifos 표준농약의 경우와 녹차추출물이 첨가된 경우에서 각각 0.01 ppm과 0.1ppm 이상의 농도에서는 농약의 농도가 높아져도 활성의 저해도에는 차이가 없었다. Fenitrothion와 methidathion는 MRL 0.2 ppm에서 1-2% AChE 활성저해를 나타내었다. 두 농약의 농도를 20 ppm까지 계속 높여감에 따라 점진적인 AChE 저해도의 증가를 나타냄으로써 농도와 저해도간의 좋은 상관관계를 나타내었다. 반면 이 두 농약에 녹차추출물이 첨가되면 모두 5 ppm부터 AChE 저해도 값에 차이가 나타나지 않았기 때문에, 농약의 농도 증가에 따른 저해도의 지속적인 상관관계가 나타나지 않았다. 이상의 결과 각 농약별로 AChE활성을 저해하는 정도가 다르며, 녹차에 의해서도 영향을 받는 저해정도가 다르다는 것을 알 수 있었다. 따라서 적절한 검사방법을 운용하기 위해서는 농약의 종류 뿐 만 아니라 각각의 시료에 따른 표준검량곡선이 요구된다. GC/MS 분석으로 표준농약과 녹차추출물이 첨가된 농약의 회수율을 비교한 결과, 90-109%의 매우 높은 회수율을 나타내었다. Most pesticidal action is the result of inhibiting acetylcholinesterase (AChE) at the nerve endings(cholinergic synapses). The residues of organophosphate pesticides in green tea were analyzed using a modified Ellman method (acetylcholinesterase inhibition assay) and GC/MS analysis. Chlorpyrifos inhibits the AChE activity directly, but not fenitrothion and methidathion. Chlorpyrifos and chlorpyrifos with green tea exhibited 75% and 83% AChE inhibition at the maximum residue limit (2 ppm), respectively. On the other hand, AChE inhibition was already saturated at 0.01 ppm and 0.1 ppm chlorpyrifos and chlorpyrifos with green tea, respectively. Fenitrothion and methidathion represented 1-2% AChE inhibition at their maximum residue limit (0.2 ppm). The AChE inhibition reaction of those pesticides was not saturated, even at 20 ppm, whereas it was saturated at 5 ppm of the pesticide with green tea. These results suggest that the AChE method requires a specific standard curve for each pesticide with agriculture products. When pesticides with green tea were analyzed by GC/MS, the recovery rate of the pesticides was 90-109%. The calibration curve revealed GC/MS to have high sensitivity and was suitable for the precise quantification of pesticides. On the other hand, the analysis takes too much time and requires highly trained technicians.

Growth retardation and differential regulation of expansin genes in chilling-stressed sweetpotato

노설아,박선희,허경혜,백경희,신정섭,배정명 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.1

We report here a first evaluation of chillingresponsive gene regulation in the sweetpotato. The growth of sweetpotato plants was severely retarded at 12C; the lengths of the leaf, petiole, and root were markedly reduced and microscopic observation revealed that the elongation growth of the epidermal cells in each of these organs was significantly reduced. We examined the transcriptional regulation of three sweetpotato expansin genes (IbEXP1, IbEXP2 and IbEXPL1) in response to various chilling temperatures (12, 16, 22, and 28C). In the leaf and petiole, the highest transcript levels were those of IbEXP1 at 28C, whereas IbEXPL1 transcript levels were highest in the root. IbEXP1 mRNA levels in the 12C-treated petiole showed a fluctuating pattern (transient decrease–recovery–stable decrease) for 48 h. In the leaf and petiole, IbEXP1 and IbEXPL1 exhibited a similar response to chilling in that their mRNA levels decreased at 22C, increased at 16C, and decreased dramatically at 12C. In contrast, mRNA levels of IbEXP2 in the leaf fell gradually as the temperature fell from 28 to 12C, while they remained unaltered in the petiole. In the root, mRNA levels of IbEXPL1 and IbEXP1 reached maximum levels at 16C, and decreased significantly at 12C. These data demonstrated that expression of these three expansin genes was ultimately down-regulated at 12C; however, transcriptional regulation of each expansin gene exhibited its own distinctive pattern in response to various chilling temperatures.

Quercetin Ameliorates Insulin Sensitivity and Liver Steatosis Partly by Increasing Adiponectin Expression in ob/ob Mice

최하늘,정수미,허경혜,김정인 한국식품과학회 2015 Food Science and Biotechnology Vol.24 No.1

The effects of quercetin on non-alcoholic fatty liver disease (NAFLD) in an obese mouse model were investigated. Five-week-old ob/ob mice were fed an AIN- 93G diet or a diet containing 0.08% quercetin, whereas ob/+ mice were provided the AIN-93G diet for 10 weeks. Quercetin significantly decreased serum glucose, triglycerides, cholesterol, and free fatty acid (FFA) levels, and homeostasis model assessment for insulin resistance (HOMA-IR) values, compared with the ob/ob control group. Quercetin lowered hepatic total lipids, triglycerides, and cholesterol levels, and the serum alanine transaminase (ALT) activities, compared with the ob/ob control group. Quercetin increased adiponectin protein expression in epididymal adipose tissue and serum adiponectin levels, and decreased serum tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1), compared with the ob/ob control group. These results indicate that quercetin could exert protective effects against development of NAFLD, partly by overexpression of adiponectin and reduction of inflammatory cytokine levels in ob/ob mice.

인삼(Panax ginseng)의 squalene synthase 유전자 발현양상 및 프로모터 분석

김태동,한정연,허경혜,최용의 한국임학회 2009 한국임학회 학술발표논문집 Vol.2009 No.-