• 한국환경농학회지
• /
• 제17권4호
• /
• pp.296-300
• /
• 1998

본 실험은 담배에서 염스트레스에 따른 생리적 반응의 연구결과(제1보)를 기초로, NaCl를 농도별로 처리하여 생체내에서 일어나는 생화학적인 변화의 구명을 통해 염해기작의 기초자료로 얻고자 수행한 결과를 요약하면 다음과 같다. 총단백질과 가용성단백질은 염농도가 높아질수록 감소하였으며 처리간에는 120mM까지는 완만히 감소하였고, 150mM에서는 급격한 감소를 나타냈다. 전기영동 패턴은 염농도의 증가에 따라 새로운 polypeptide band의 생성과 소멸은 없었으며 약 74Kd의 polypeptide band에서 30mM과 60mM까지는 뚜렷한 양이 증가하였고 90mM부터는 감소하였다. 엽록소함량은 염농도의 증가에 따라 감소하였으며 특히 염해에 의한 반응은 엽록소b보다는 엽록소a가 민감하였다. ATPase활성과 peroxidase의 활성은 염농도가 높아질수록 120mM까지는 일정하게 증가하였으나 150mM에서 급격히 증가하여 담배의 염해의 생화학적인 제한범위는 120mM로 나타났다.

• Journal of Microbiology and Biotechnology
• /
• 제23권8호
• /
• pp.1047-1054
• /
• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• Current Research on Agriculture and Life Sciences
• /
• 제17권
• /
• pp.15-20
• /
• 1999

엽록제 small HSP의 기능을 조사하기 위하여 항상적으로 발현하는 형질전환 식물체를 작성하였다. 고온 스트레스 하에서의 형질전환 식물체의 고온내성을 chlorophyll 형광으로 측정하였다. Leaf disc를 고온조건에서 5분간 처리한 후, 광화학계 II의 불활성화를 나타내는 Fo 값의 증가 또는 Fv 값의 감소치를 조사하였다. 형질전환 식물체는 고온 스트레스 하에서의 이들 값의 증감율이 현격히 감소하였다. 또한 유식물체를 $52^{\circ}C$에서 45분간 처리한 후, $25^{\circ}C$에서 계속적으로 배양하였을 때, 비형질전환 식물체는 전부 고사하였으나, 형질전환 식물체의 약 80%는 생존하였다. 이러한 결과는 엽록체 small HSP가 고온 스트레스 하에서 광합성기구를 보호하는데 있어서 중요한 기능을 담당하고 있음을 나타낸다.

• Applied Biological Chemistry
• /
• 제40권4호
• /
• pp.271-276
• /
• 1997

박테리오파아지 T7 RNA polymerase 유전자를 식물체내에서 이용할 수 있을지 알아보기 위하여 상처유발인 감자 단백질 분해효소 억제제 유전자의 프로모터에 박테리오파지 T7 RNA polymerase 유전자를 연결시킨 후 담배에 도입시켰다. 형질전환 식물체의 DNA에 대한 Southern hybridization에 의하면 T7 RNA polymerase 유전자가 식물체내에 1-2 copy가 존재하며, Northern hybridization에 의하면 T7 RNA polymerase의 RNA가 상처에 따라 생성되는 것을 확인하였다. 또한 Western hybridization에 의하면 식물체내 T7 RNA polymerase 단백질이 생성되는데 그 크기는 대장균에서 생성되는 단백질 크기와 유사한 80 kDa 이었으며 시험관내에서 전사체에 뉴클레오타이드를 결합시키는 능력이 있음도 확인하였다. 따라서 T7 RNA polymerase 유전자를 이용하여 식물체내에서 원하는 유전자의 발현을 증대시킬 수 있을 것으로 사료된다.

• 한국연초학회지
• /
• 제16권2호
• /
• pp.144-151
• /
• 1994

Using a flue-cured tobacco variety, BU 109, effect of growth regulators(Fatty Alcohol and C-MH) on the change of invertase activity and sugar metabolites were investigated. Invertase activity in untreated leaf tissue was decreased along with maturation of leaf, However, a momentary increase of the activity was observed in leaves by the dual treatment of fatty alcohol and C-MH regardless of leaf position while sole C-MH treatment resulted in activity increase by 14 days after the treatment. Similar tendency was observed in stalk. Sugar content in leaf was increased immediately after the treatment but no significant increase at large resulted until 14 days after treatment. After harvest, reducing sugar was increased by the growth regulators. Nevertheless, in case of dual treatment, the total sugar content was not different with that of untreated control. R/T ratio was gradually increased after topping stage and reached maximum at 14 days after treatment of growth regulators. It decreased a little after harvest but the RR ratio was relatively higher due to increase of reducing sugar resulted by the treatment of the chemicals. Upon treatment of growth regulators, reducing sugar content was increased in lugs and leaves compared to untreated control and the content of sugar metabolites was increased by the use of the chemicals either at lower or higher than recommended dose. Key words : MH, FA, invertase activity, total sugar, reducing sugar

• 한국연초학회지
• /
• 제19권2호
• /
• pp.92-101
• /
• 1997

This study was conducted to investigate the effect of fly ash on the yield and quality and to determine the optimum application amount of fly ash for tobacco(Nicotiana tabacum L). Two kinds of fly ash, anthracite and bituminous coal, were treated with different levels of 0, 20, 40, 60 MT/ha. Dry weights of tobacco at middle and topping growth stage were increased with application of fly ash, showing the highest dry weight at 40 MT/ha in both kinds of fly ash. It was showed that the bituminous coal had a little more effective for yield than that of anthracite. Comparing with the control, yields of tobacco applied with fly ash were significantly increased about 17.7% and 17.1% by the application of bituminous coal and anthracite, respectively. Quality of flue-cured leaves was better by application of fly ash than that of the control. The quality index was given the highest at 40 MT/ha for bituminous coal increasing by 24.6% and at 60 MT/ha fur anthracite increasing by 13.4% compared with the control. The economical efficiency considered of the yield and quality of tobacco was the highest at 40 MT/ha of bituminous. Soil pH, contents of available P2O5, organic matter, exchangeable Ca2+ and Mg2+ of soil during the growing season were increased by application of fly ash, showing more effectiveness in bituminous than that in anthracite. By the application of fly ash, the nutrients availability and the acidity of soil were reformed and they caused significantly the increase of growths yield, and quality of tobacco. By the application of lime reforming soil acidity, growth response, yields and quality of tobacco were not increased compared to the control, although the effect of reforming soil pH was remarkable.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
• /
• pp.425-428
• /
• 2000

본 연구에서는 재조합단백질 생산을 위한 식물세포배양용 세포배양기 운전에서 배지교환식 배양과 기체조성에 따른 영향을 관찰하였다. 배지교환식 배양에서는 6일째에 배지교환을 시작하는 것이 세포의 생장을 저해하지 않았으며 교환용 배지의 조성을 120 g/L sucrose와 2배 농축된 염농도를 하였을 때 가장 높은 세포농도인 20.0 gDCW/L를 나타내었으며 GUS 생산량은 60 g/L sucrose와 기본배지와 같은 농도의 염농도로 배지를 교환했을 때 12.9 U/mL로 다른 경우에 비하여 $1.5^{\sim}4.6$ 배 증가된 결과를 보였다. 그리고 $CO_2$의 농도를 5%로 높인 공기로 통기했을 때는 세포의 초기생장을 증진시켰으며 0.2 vvm의 통기량에서 5% $CO_2$로 통기시킨 경우는 일반 공기를 0.1 vvm으로 통기시킨 경우보다도 77% 높은 GUS의 생산량을 나타내었다.

• KSBB Journal
• /
• 제30권2호
• /
• pp.82-90
• /
• 2015

Plant cell immobilization can protect plant cells from shear forces and increase the stability of gene. An additional advantage of immobilization is the easiness for performing continuous culture with cell recycling. Therefore plant cell immobilization can overcome the limitations of plant cell applications. In addition, target protein should be selected from pharmaceutical proteins to get rid of low expression level problem. The enhanced production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in immobilized Nicotiana tabacum suspension cell cultures. When the cells were immobilized in polyurethane foam, specific production of hGM-CSF was higher than that in alginate bead immobilization. Optimum continuous culture condition was the addition of 60 g/L sucrose in growth media with exchanging media every 6 day. Under the same condition, specific hGM-CSF production was 7 times higher in a 500-mL spinner flask than that in 100-mL Erlenmeyer flasks. Therefore, development of an effective immobilization process would be possible when the advantage of easy cell recycling was used. Consequently, enhanced production of target proteins could be possible in immobilized continuous cultures when the advantages of immobilization were applied.

• BMB Reports
• /
• 제38권5호
• /
• pp.595-601
• /
• 2005

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7% and 61.9% identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2% identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both $Ca^{2+}$ and calmodulin. The GAD-encoded 56~58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a $Ca^{2+}$/calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

• 식물조직배양학회지
• /
• 제21권2호
• /
• pp.99-103
• /
• 1994

비선택성 제초제인 bialaphos는 고등식물에 있어서 glutamine 합성을 억제하여 식물체를 고사 시키는 능력을 갖고 있다. 본 연구에서 acetylteansferase에 의해 encoding된 bialaphos 저항성 유전자(bar gene)는 세균(Pseudomonas sryngae pv tabaci)의 genomic DNA로부터 cloning된 것을 사용하였다. 수분시킨 담배의 화계에 일정한 시간별로 bar 유전자를 처리한 결과 수분 후 30-40시간 사이의 처리구 에서 형질전환 식물체가 가장 많이 얻어 졌다. 그러한 형질전환 식물체의 kanamycin과 bialaphos 저항성 형질은 자식후대(T$_1$, T$_2$)에 있어서도 우성형질로 유전되었으나 wild type의 담배는 상기의 약제를 처리 하였을때 전부 고사하였다. 그리고, T$_1$세대의 형질전환 식물체로부터 전 염색체 DNA를 추출하여 Southern 분석한 결과 bar 유전자가 식물의 염색체상에 안정하게 존재하는 것을 확인하였다. 이상의 결과로부터 담배의 수분, 수정 시기에 외부유전자인 bar를 화주에 처리함으로써 bialaphos 저항성 식물을 만들어낼 수 있었다.