• KSBB Journal
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• 제18권6호
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• pp.435-439
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• 2003

형질 전환된 담배 seed에서 담배 식물체를 유도하여 White 액체 배지에서 기관 배양하였다. 암조건, sucrose 2%의 조건에서 좋은 growth pattern을 나타내었고, 계대 배양은 외마디법을 이용하여 2주마다 하였다. 기관 배양에서 hGM-CSF production pattern을 보면, intracellular에서는 큰 변화 없이 약 30 ng/g의 일정한 농도를 나타내었다. Extracellular에서 hGM-CSF 농도는 배양 6일 이후부터 급속하게 증가하기 시작하여 배양 12일째에 약 0.2ng/$m\ell$의 농도를 나타낸다. 기관배양은 다른 식물세포 배양 시스템에 비해 생산되어진 단백질의 안정성이 크다는 장점에 비해 세포 내에서 배지 내로의 단백질 분비가 적다는 단점이 있다. 이를 극복하기 위해 다양한 permeabilizing agents를 투여하여 담배 세포의 permeability를 증가시키고자 하였다. 그 결과, Pluronic F-68과 PEG8000을 첨가한 경우 담배 세포에서 배지 내로의 단백질 분비가 원활해졌음을 확인할 수 있었다.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 춘계학술발표대회:발표논문요지록
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• pp.18-18
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• 1999

We characterized a cDNA clone for a low molecular weight heat-shock protein (LMW HSP) from tobacco named TLHS-l. Nucleotide sequence determination of TLHS-1 identified an open reading frame for 159 amino acids. To the upstream of the open reading frame, a sequence of 124 nucleotides was determined. To the 3' downstream of the open reading frame, 212 nucleotides were identified which carried poly(A)-tail. Comparison of the open reading frame and hydropathy plot of TLHS-1 with the previously reported class I LMW HSPs showed high identity which classified TLHS-1 as a class I LMW HSP cDNA clone. We proposed that there are six consensus regions in class I LMW HSPs. RNA blot hybridization for TLHS-1 showed a typical expression pattern of heat-shock-inducible gene from three common tobacco cultivars. The open reading frame of TLHS-1 was overexpressed in Escherichia coli. TLHS-1 protein confers thermal protection of other proteins in vitro and in vivo. Thermal induced aggregation of citrate synthase was reduced by purified TLHS-1 protein, and thermal death rate at $50^{\circ}C$ was reduced in E. coli expressing TLHS-l. From these data, we can expect that TLHS-1 acts as a molecular chaperone.perone.

• 한국자원식물학회지
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• 제11권2호
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• pp.188-194
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• 1998

This experiment was conducted to introduce phosphinothricin acetyl -transferase(PAT) gene, resistant to basta and non-selective herbidide, into tobacco(Nicotiana tabacum cv.BY4). For shoot formation,tobacco leaf disks were placed on the MS medium supplemented with 2.0mg/L BA and 0.1mg/L NAA. In this medium condition, tobacco leaf disces were cocultivated with A. tumefaciens MP90 containing NPT IIand PAT resistant to kanamycin and Basta, respectively. Shoots were obtained in the medium containing antibiotics, and those were transferred to rooting medium supplemented with 0.1mg/L NAA and antibiotics. The plants obtaining roots were transplanted into soil. Phenotype of transgenic tobacco plant was mostly as normal plant. However, about 5% was abnormal plant, which did not set seeds. PCR analysis and southern blot were performed to determine transformation. As the results, it was confirmed that PAT gene was stably integrated into tobacco genome.When herbicide, basta, was sprayed to the plants confirmed by PCR, the transgenic plants showed normal growth, whereas normal plants died. Therefore, the result of this experiment show that tobacco transformation for the resistance to basta, non-selective herbicide, was successful because PAT gene was stably integrated into tobacco.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.79-84
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• 1999

Oxidative stress is one of the major environmental stresses to plants. Reactive oxygen species (ROS) generated during metabolic processes damage cellular functions and consequently lead to cell death. Fortunately plants have in vivo defense system by which the ROS is scavenged by enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). In attempts to understand the protection mechanism of plant against oxidative stress, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plansts thet expressed both SOD and APX in chloroplast using Agrobacterum-mediated transformation and evaluated their protection capabilities against methyl viologen (MV, paraquat) -mediated oxidative damage. Three double transformants (CAI, CA2, and CA3) expressed the chimeric CuZnSOD and chimeric APX in chloroplast, and one transformant (AM) expressed the chimeric APX and chimeric MnSOD in chloroplast. In addition, we obtained three lines of transformants (C/Al, C/A2, and A/C) that expressed the APX and SOD than control plants, and more resistant to oxidative stress caused by MV. TRansformants (C/A and A/C) overexpressing MnSOD, CuZnSOD and APX at the same time showed the highest resistance to MV-mediated oxidative stress among the transformants.

• 한국연초학회지
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• 제18권2호
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• pp.138-144
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• 1996

This experiment was conducted to obtain the basic information on the morphological and physiological changes in tobacco leaf during the growth period by measuring the changes of chlorophyll, sugar, lipid and mineral contents in tobacco plant. Leaf length and width have been fully developed at 25 days after leaf emergence. Dry weight was rapidly increased between 10 and 15 days after leaf emergence and reached the highest at 30 days. Crude lipid content, palmitic acid, and the major saturated fatty acid were increased with progressing senescence, while unsaturated fatty acid including linolenic acid was decreased as the senescence was advanced. The total nitrogen content showed the highest value at IS days after leaf emergence. On the other hand, the total sugar content showed the highest value at 45 days after leaf emergence and glucose, fructose and sucrose were decreased with leaf development and increased at the end of senescence. The content of chlorophyll showed the highest value at 15 days after leaf emergence and began to decrease at 30 days after leaf emergence. The contents of p, Cu, Zn, and Fe in tobacco leaves were decreased by the end of senescence after leaf emergence but those of Ca, Mg, and Mn in leaves were increased. Key words : Nicotiana tabacum chlorophyll, fatty acid, senescence.

• 한국연초학회지
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• 제19권1호
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• pp.57-63
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• 1997

The composition of leaf surface lipid in Flue-cured tobacco and their changes during curing was investigated. The flue-cured variety, Nicotiana tabacum cv. NC 82 was cultivated at Eumsung experiment station in 1996. The samples of riced leaves with different stalk Position(Lugs, Cutter, Leaf and Tips) and different curing stage at half yellowing(24hr), yellowing(48 hr) , color axing(72 hr), midrib drying(96 hr) and cured(120 hr) were collected for analysis of leaf surface lipid. $\alpha$ - and $\beta$ - 4, 8, 13-Duvatriene-1, 3-diol($\alpha$, $\beta$-DVT) were major components in leaf surface lipid extracted with methylene chloride and sugar ester was detected slightly DVT content was increased with ascending stalk position, but increasing trend of total hydrocarbon was not observed. While DVT was decreased throughout curing of tobacco leaves, hydrocarbon content did not show significant change during curing process. Twenty-two duvane compounds were detected by capillary GC in duvane fraction isolated from leaf surface lipid and of which 11 compounds were identified by GC-MS. These compounds were decreased with curing in all stalk position.

• 한국연초학회지
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• 제19권1호
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• pp.11-17
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• 1997

A flue-cured tobacco variety (Nicotiana tabacum cv. Wisconsin) was used for Plant transformation with the complementary DNA (cDNA) of potato virus Y-necrosis strain (PVY-VN) replicase gone (Nb) which was synthesized through reverse-transcription Primed with oligo(dT) and Polymerization using RNase H-digested template. The cDNA was cloned into Plant expression vector Plasmid (PMBP2), and introduced into tobacco plants by co-culturing tobacco leaf disks with Agrobacterium tumefaciens LBA4404 containing the plasmid before Plant regeneration. Eight Plants, in which the inserted cDNA fragment was detected by Polymerase chain reaction (PCR), out of 70 putative transformants inserted with sense-oriented Mb cDNA showed no symptom at 3 weeks after inoculation, while the other 62 plants, and all plants with vector gone only and antisense-oriented NIb cDNA had susceptible vein-necrosis symptoms. However, only 2 of the 8 resistant plants were highly resistant, which remained symptomless up to 10 weeks after inoculation. Among the first progenies (T1) from self-fertilized seeds of the two resistant transgenic plants, less than 10 % of 71 plants appeared highly resistant (with no symptom), 70% moderately resistant (with mild symptoms on 1 - 2 leaves), and about 20% susceptible (with susceptible symptoms on 3 or more leaves) at 3 weeks after inoculation. These results suggest that the PVY resistance was inherited in the 71 generation. Key words : potato virus Y. viral replicase gene, transgenic tobacco Plants, resistance.

• 한국연초학회지
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• 제15권2호
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• pp.145-151
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• 1993

Using a flue - cured tobacco variety, KF lH9, recently bred and released to farming, effect of growth regulators(fatty alcohol and C - MH) on the change of alkaloid contents and dynamics of MH uptake and distribution were investigated. An efficient sucker control method was also pursued. Treatment of fatty alcohol at button stage remarkably increased the rate of C - MH uptake. However, except in the plants treated with 2- fold volume of C - MH, residue of C -MH was kept under maximum residue level(80 ppd). C - MH residue in stalk was higher than in foliage in general. Compared to untreated control, leaf weight per unit area was increased by the treatment even in lower leaves where vigorous transfer of C - MH from treated tips was indicated and sucker control higher than 94.9% was achieved with all treatments except for sole half volume C - MH treatment at topping stage. Alkaloid content in foliage was reduced by dual treatment of fatty alcohol and C - MH but increased in stalk and root.

• 한국연초학회지
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• 제15권2호
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• pp.152-160
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• 1993

Using a flue- cured tobacco variety, KF 109, effect of growth regulators(fatty alcohol and C- MH) on the change of protein, DNA, and RNA were investigated. Generally, inhibition of DNA synthesis was observed soon after become notably reduced when checked on 14 days after the treatment. Fatty alcohol treatment appeared to alleviate the inhibition of DNA synthesis caused by the C - MH treatment. It was also observed that in the tips DNA content increased slightly at the early stage after the C - MH treatment but evident reduction of it was resulted from 7th day after the treatment. RNA content in cutters and tips was increased initially but variable transcription inhibitory activities - not so obvious as was observed in DNA synthesis - according to leaf positions were shown thereafter. Ripening of leaves probably due to senescence was advanced by the treatment of the growth regulators. DNA content in root was relatively higher in plants treated with the growth regulators while it was clearly decreased in stalk, However, RNA contents in tissue of stalk and root was not different with that of foliage. Increase of protein content in foliage as well as in stalk was evident 14 days after dual treatment of fatty alcohol and C - MH.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권3호
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• pp.187-191
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• 2003

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium. with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. compared to the controls (4.72 $\mu\textrm{g}$/L), the extracellular hGM-CSF level could be increased to 39.78 $\mu\textrm{g}$/L with the addition of 5 g/L of gelatin.