패랭이꽃속 Dianthus gratianopol의 현탁배양세포로부터 Shoot 증식과 식물체 재분화

김준철,Kim Joon-Chul 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.4

패랭이꽃속 Dianthus gratianopol 잎절편체로부터 캘러스를 유기하여 표면에 광택이 있고 치밀하게 배열되고 연한 녹색의 shoot-forming 캘러스를 선별하였다. 유기된 캘러스를 잘게 자르고 1.0 mg/L 2,4-D와 0.5 mg/L BAP가 포함된 CP 액체배지에서 7일 간격으로 계대배양하여 균일한 SF형태의 조직이 치밀한 세포괴를 얻을 수 있었고 이들 세포괴를 1.0 mg/L 2,4-D와 0.5 mg/L BAP, 1.5 mg/L 2,4-D와 0.5 mg/L BAP의 MS고체배지에서 배양했을 때 재분화능력이 있는 캘러스를 유도할 수 있었다. 이들 캘러스는 1.0mg/L TDZ과 0.5 mg/L PAA가 포함된 MS재분화배지에 치상하였더니 녹화되기 시작하여 배양 4주후부터는 다량의shoot 원기가 형성되고 shoot증식이 가능하였으며 평균 87%의 shoot 형성률을 보였다. 배양 9주후 절단된 shoot는 0.1 mg/L NAA가 첨가된 배지에서 뿌리를 형성하여 화뢰와 꽃을 갖고 있는 완전한 식물체로 재분화되었다. 또한 재분화 능력이 있는 캘러스 정단 원기를 절단한 절편체는 1.0 mg/L TDZ 과 0.5 mg/L PAA가 포함된 MS재분화 배지에서 직접 부정아를 형성하였고 이들 부정아로부터 다량의 shoot 증식이 가능하여 식물체를 효과적으로 얻을 수 있었다. Conditions for efficient organogenesis and plant regeneration from Dianthus gratianopol suspension cultured cells were established. Shoot-forming calli of glossy surface, pale green and knobby type were selected from leaf explant-derived calli and were suspension-subcultured every week in CP liquid medium with 1.0 mg/L 2,4-D and 0.5 mg/L BAP. Combinations of 1.0 mg/L 2,4-D and 0.5 mg/L BAP, and 1.5 mg/L 2,4-D and 0.5 mg/L BAP were effective for the induction of regenerative callus from the suspension cultured cell clusters. Multiple shoot primordia were initiated from the green spots of these regenerative callus and formed shoots on MS medium with 1.0 mg/L TDZ and 0.5 mg/L PAA. Shoot regeneration frequency (calli regenerating at least one shoot) was about 87%. For plant regeneration, proliferated shoots were excised and transferred to MS medium with 0.1 mg/L NAA for root initiation after 9 weeks of culture. The regenerants were potted in soil and formed the flowering buds and petals. Also, adventitious shoots were formed from the excised green shoot primordia of regenerative callus and these shoots proliferated successfully and regenerated to whole plants.

전단 스트레스에 의한 심방근세포 커넥신 해미채널의 활성화

김준철(Joon-Chul Kim),우선희(Sun-Hee Woo) 대한약학회 2019 약학회지 Vol.63 No.4

Abstract Shear stress induces global Ca waves in atrial myocytes and this Ca wave has been shown to be sensitive to connexin (Cx) channel interventions. In this study, we investigated whether shear stress activates Cx by measuring dye flux through the Cx channels in rat atrial myocytes. Because the pores of the hemichannels are large enough to permit flux of large molecules up to 1000 MW, we used a Cx channel-permeable dye, Calcein, and a Cx-impermeable dye, Oregon to evaluate Cx-specific activity under shear stress. Application of shear stress (~16 dyn/cm 2 ) using micro-jet apparatus to single atrial cells induced a significant increase in cellular Calcein efflux. The Calcein efflux was largely potentiated by the use of Ca -free external solutions which enhance Cx hemichannel activity. However, it was suppressed by 95% following the pre-treatment of La (2 mM), a gap junction blocker. Pre-exposure to the pannexin inhibitor probenecid (800 mM) did not alter the calcein efflux under shear stress. In contrast, the Oregon signal was not significantly affected by shear stress exposure, suggesting that calcein efflux by shear exposure was not due to nonspecific increase in membrane permeability. In a monolayer of HL-1 atrial cell culture treated by the external solutions containing Cx-permeable Lucifer Yellow dye, cellular dye influx was increased by shear stress. These results suggest that shear stress activates Cx hemichannels in atrial myocytes.

스테로이드 유도기작에 의한 애기장대 배주발달에서 BEL1 의 기능 분석

김준철(Joon-Chul Kim) 강원대학교 기초과학연구소 2005 기초과학연구 Vol.16 No.-

  애기장대 (Arabidopsis thaliana)의 방사대칭형 배주원기는 초기배반조직에서 자라기 시작하여 proximal, 중앙, distal 부위로 구분되었고 배주원기의 중앙부위의 원형조직으로부터 내주피가 형성되었고 이어 외주피가 형성되었다. 외주피의 형성은 처음에는 배주의 배축면(abaxial) 쪽에서 다음에는 향축면(adaxial)쪽까지 이어져 주심과 내주피를 덮었다. 외주피는 배주원기의 배축면 쪽의 형성에 주로 참여하여 비대칭적으로 자랐다.   배주발달에서 높게 발현되는 BELL 단백질의 기능을 조사하기 위해 스테로이드에 의해 유도되는 BEL1::GR 벡터를 구성하였고 암컷불임이고 주피형성이 되지 않는 bel 돌연변이에 삽입하여 배주발달에서 전사활성의 시기와 양을 조절을 조사하였다. Kanamycin 배지에서 야생형으로부터 35S::BEL1::GR 형질전환system을 이용하여 20개의 형질전환체와 bel 돌연변이에서 36 계통의 형질전환체를 선발하였고 이들 표현형은 야생형과 큰 차이를 보이지 않아 BEL1 활성이 전사후 조절에 기인하는 것으로 생각된다. DEX를 처리한 35S::BELL::GR bel 형질전환체는 임성을 회복하여 종자를 형성하였고 이는 BELL-GR-DEX로 결합되어 핵 내로 이동시켜 이동한 BELL의 활성에 의해 배주발달 후기에서 배축면 주피의 형성을 촉진하여 주심을 덮는 것으로 판단된다.   이상의 결과로부터 초기배주 원기는 3부분, distal, 중앙, proximal으로 발달되고 이시기에서 BELL 기능은 배주 내 극성을 유지하여 주피 분화를 촉진하는 것으로 추정된다.   As radically symmetrical ovule primordia grew, three distinct proximal-distal pattern elements were formed in Arabidopsis ovule development. The inner integument initiated from a ring of tissue in the central pattern element of each primordium. This was closely followed by initiation of the outer integument, first on the abaxial side of the ovule and next on the adaxial side of the ovule. The outer integument covered both the nucellus and inner integument. The outer integument grew asymmetrically, favouring the abaxial side of the ovule primordium.   To further examine the role of BEL1 homeodomain protein highly expressed in developing ovules, We constructed a steroid-inducible BEL1 :: GR fusion protein to control the amount and timing of the transcription factor activity in developing bel mutant ovules which were female-sterile and failed to form integuments. Twenty transformants from wild-type plants were selected on kanamycin plates. Induction of ectopic BEL1 activity in wild-type plants caused no obvious phenotype. We also made 35S::BEL1::GR transgene plants from the bel mutant plants and found that there was no obvious phenotype in thirty six bell transformant lines.   DEX spraying-treatment on the inflorescences of the 35S::BEL1::GR bel transgene plants complemented and rescued the bell infertility and these transgene plants gave the siliques with seeds. Induction of BEL1 activity late in ovule development promoted the formation of abaxialized integuments.   We suggest the BEL1 function in ovule development that BEL1 establishes and maintains polarity in the ovule, and promotes the integument differentiation.

한국의 성결교파들 : 구세군 안에 있는 메소디즘 -구세군 역사와 교리 배경을 중심으로

김준철 ( Joon Chul Kim ) 현대기독교역사연구소 2001 성결교회와 신학 Vol.5 No.-

참나물(Pimpinella brachycarpa)의 배발생 관련 PbMYB56의 특성

김준철(Joon-Chul Kim) 강원대학교 기초과학연구소 2005 기초과학연구 Vol.16 No.-

  참나물(Pimpinella brachycarpa)의 shoot-tip 절편체로부터 체세포배발생캘러스를 선발하여 현탁배양하였으며 이들 배양세포괴들로부터 유래된 배발생캘러스는 연한 노란색을 띠며 체세포배로 분화되었다. 체세포배는 배양기간이 계속되면 발아되어 어린식물체로 되었으며 식물체 재분화는 암조건에서 초기 2주간 배양했을 때 다소 양호하였으나, 이 후 배양에서는 24 E m 2sec 1의 연속광 하에서 배양했을때 체세포배로부터 식물체로 재분화 빈도 및 식물체 수는 더욱 높았다. 참나물의 체세포배로부터 발아 및 식물체의 재분화에는 0.6 M GA3에서 비교적 용이하게 발아되어 참나물 체세포배의 발생경로를 통해 번식이 가능하였다. 참나물 배발생캘러스로부터 분리한 mRNA로부터 cDNA library를 합성하여 PCR을 수행한 결과 cDNA library로부터 배발생 callus 유래 PbMYB56를 cloning하였다. PbMYB56-cDNA sequence를 토대로 제작된 pMYB56-1(5"-gaaatggctgcaataaaatc-3"), pMYB56-2(5"-cgaggttttgaataatggagac-3"), pMYB56-3 (gaaatggctgcaataaaatc-3")를 이용하여 amplification을 하여 약 2356 bp의 PCR product를 얻었다. 이후 pGEM T-easy vector (pGEM-T and pGEM-T Easy Vector Systems ; Promega)에 cloning하여 양방향으로 Automatic sequencing (ALF express)을 하여 기존의 cDNA sequence와 비교 한 개의 1200 bp 크기의 intron 부위를 찾을 수 있었다. 참나물(Pimpinella Brachycapa)에서의 Pbmyb gene은 다소 낮은 copy 수인 약 4개의 copy 수를 가지고 있음을 알 수 있었다. 식물의 developmental stage의 단계별 PbMYB56의 expression pattern 과 expression quantity는 분화된 plant stage에서 가장 많이 발현 되었고 다음으로 shoot와 callus stage에서 발현되는 것을 알 수 있었다. 그러나 callus stage에서는 RT-PCR에 비해 northern hybridization에서 signal이 매우 약해 PbMYB56이 in vitro 분화과정에서 배 발생에 관여하는 specific한 유전자의 기능보다는 transcription regulator로 작용하는 MYB protein으로서 tissue specific gene일 가능성을 보여 주었다.   Somatic embryogenic calli from shoot-tip explants of Pimpinella brachycarpa were suspension-cultured and selected embryogenic calli were transferred to MS medium containing 0.2 M 2,4-D, 0.5 M BAP and 10.0 M TDZ. Embryogenic calli expanded three-to four-fold and globular embryos were appeared on the surface of a pale-yellow calli grown on MS media containing 10.0 M TDZ only, or in combination with 0.5 M NAA. The embryos seperated from the callus and placed in the liquid MS medium containing 0.6 uM GA3 without 0.2% gelrite. Fully germinated embryos matured into plantlets within 2-3 weeks. Normal plants developed from the somatic embryos and grew forming multiple shoots. To amplify PbMYB56-cDNA sequence from embryogenic callus of Pimpinella brachycarpa, the oligonucleotide primers were syntheisised as primers of pMYB56-1 (5"-gaaatggctgcaataaaatc-3"), pMYB56-2(5"-cgaggttttgaataatggggagac-3"), pMYB56-3 (gaaatggctgcaataaaatc-3"). PbMYB56 was 2356 bp and contained an intron of 1200 bp. PbMYB56 gene had four copies as low copy number. In the developmental stage the expression pattern and expression quantity of Pbmyb gene by RT-PCR showed highly in a whole plant stage with petioles and leaves. Also, expressed in shoots but lowly in embryogenic callus stage. we suggest that this PbMYB56 gene may transcriptionally be regulated and expressed for tissue specific function rather than embryo specific function.

유체 압력에 의한 흰쥐 심실근세포 pH의 감소 및 Na+ -H+ 교환체의 활성화

김준철(Joon-Chul Kim),우선희(Sun-Hee Woo) 대한약학회 2011 약학회지 Vol.55 No.3

An increase in ventricular pressure can alter cardiac excitation and contraction. Recent report has demonstrated that fluid pressure (FP) suppresses L-type Ca2+ current with acceleration of the current inactivation in ventricular myocytes. Since the L-type Ca2+ channels known to be regulated by intracellular pH (pHi), this study was designed to explore whether pressurized fluid flow affects pHi in isolated rat ventricular myocytes. A flow of pressurized (~16 dyne/cm2) fluid, identical to that bathing the myocytes, was applied onto single myocytes, and intracellular H+ concentration was monitored using confocal H+ imaging. FP significantly decreased pHi by 0.07±0.01 pH units (n=16, P<0.01). Intracellular acidosis enhances the activity of Na+-H+ exchanger (NHE). Therefore, we examined if the NHE activity is increased by FP using the NHE inhibitor, HOE642. Although HOE642 did not alter pHi in control conditions, it decreased pHi in cells pre-exposed to FP, suggesting enhancement of NHE activity by FP. In addition, FP-induced intracellular acidosis was larger in cells pre-treated with HOE642 than in cells under the control conditions. These results suggest that FP induces intracellular acidosis and that NHE may contribute to extrude H+ during the FP-induced acidosis in rat ventricular myocytes.

연초의 (煙草) Callus , 액체배양세포 및 엽조직의 Peroxidase Isozyme 과 활성에 관하여

김준철(Joon Chul Kim),윤경은(Kyung Eun Yoon),이광웅(Kwang Woong Lee) 한국식물학회 1981 Journal of Plant Biology Vol.24 No.2

The present researches were carried out to investigate the relation between growth and peroxidase. The activities and isozyme patterns of peroxidase in the callus, suspension-cultured cells and leaves of tobacco plant (Nicotiana tabacum L. cv. Virginia 115) were subjected for the purpose. The differences in the growth and isozyme patterns were noticed among the cells cultured in Linsmaier-Skoog meida supplemented with 3 kinds of plant growth regulators-kinetin, 2,4-D and α-NAA. In cells cultured in the media with α-NAA or 2,4-D, the growth was promoted, while the numbers of isozyme and activities of peroxidase were decreased. A fast migrating isozyme was observed in the cells grown in the medium with α-NAA. The older leaves grew, the more numbers and activities of the enzyme in tobacco plant were observed. It was assumed that number of isozymes and activity of the peroxidase were low in the callus, cell and leaf, when they were growing actively.

Nicotiana tabacum 과 Petunia inflata 의 전기적 원형질체융합에 의한 속간 (屬間) 체세포 유종의 (維種) 생성

김준철(Joon Chul Kim),이석구(Suk Koo Lee),이광웅(Kwang Woong Lee) 한국식물학회 1987 Journal of Plant Biology Vol.30 No.1

Leaf mesophyll protoplasts of Nicotiana tabacum (nitrate reductase deficient mutant) were fused with cell suspension protoplasts of albino Petunia inflata in an electric field. Hybrid cell colonies were selected for nitrate reductase proficiency and chlorophyll synthesis. Five hybrid plant lines, regenerated from the selected calli lines, were analysed by electrophoresis, number of chromosomes and morphological characters. Somatic hybrid plants showed both parent patterns in the isozymes of isoleucine aminopeptidase and esterase. The hybrids had the expected chromosome number of 62 and exhibited an intermediate floral morphology when compared with the parents, but plant height and leaf arrangement were similar to N. tabacum.

Nicotiana tabacum 과 N. glutinosa 간 원형질체융합 식물체에 있어서 엽록체 DNA 의 제한효소단편의 (制限酵素斷片) 유형

김준철(Joon Chul Kim),최성진(Seong Jin Choi) 한국식물학회 1991 Journal of Plant Biology Vol.34 No.1

Mesophyll protoplasts of Nicotiana tabacum (NR^-/SR^+) and N. glutinosa were electrofused with AC field of 0.5 MHz and 1 kV DC pulse for 2 ms. Fused protoplasts were selected and cultured to the green cell clusters in MSNO_3 medium containing 1.2 ㎍/㎖ streptomycin sulfate. Four plant lines regenerated from selected colonies showed both parental morphological characteristics of leaf and flower and these plant lines were confirmed as somatic hybrids based on electrophoretic patterns of leaf peroxidase. In XhoI restriction patterns of chloroplast DNA, these hybrid plant lines expressed both parent common restriction sites and parent specific sites. One of these hybrid lines exhibited interspecific pattern of both parental chloroplast genomes, indicating nine both parent common sites, one N. tabacum specific site and two N. glutinosa specific sites.

근치적 전립선적출술 후 요자제의 진정한 의미는?

김준철(Joon Chul Kim) 대한비뇨기종양학회 2007 대한비뇨기종양학회지 Vol.5 No.1