• Journal of Plant Biotechnology
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• 제47권2호
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• pp.157-163
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• 2020

급속한 산업화와 인구증가에 따른 심각한 환경과 식량문제는 인류의 생존을 위협하는 가장 중요한 현안문제로 대두되고 있다. 또한, 인구 증가에 따른 식량부족을 해결하기 위하여 농약과 화학비료의 무분별한 사용으로 인하여 농토는 산성화되어 황폐화가 되고 있고 먹이사슬 및 자연생태계의 파괴는 더 많은 농약의 사용을 필요하고 있다. 과다한 농약 사용으로 경제적인 부담의 가중과 잔류 농약으로 인한 소비자의 건강을 위협하고 있다. 또한, 증가된 화석연료의 사용은 공기중의 이산화탄소를 증가시켜 지구 온도의 상승을 초래하고 있으며, 결과적으로 기상이변, 지구온난화 및 사막화 등의 심각한 환경문제를 초래했다. 특히, 서늘한 기온에서 잘 자라는 배추, 무우, 감자 등의 고령지 농작물의 질적인 저하를 초래하여 피해가 증가하고 있지만, 최근들어 감자와 같은 알카리성 건강식품의 붐으로 수요가 증가되고 있다. 본 연구에서 환경스트레스에 관여하는 대두의 특이적인 칼모듈린, GmCaM-4 유전자를 감자에 과발현 시켜서 PR 유전자들의 발현이 지속적으로 유지되어 식물방어 기작이 활성화 되었음을 확인하였다. 또한, 그 형질전환 식물체는 초민감성 세포사멸 현상을 보였고, 무름병을 일으키는 병원균인 Erwinia carotovora subsp. Carotovora (Ecc)를 이용하여 GmCaM-4가 과발현된 감자에서 병 저항성이 증가하는 것을 확인 하였다. 최종적으로 지금까지 많이 연구되고 보고된 유전자원인 대두의 GmCaM-4 유전자를 활용하여 주요 식량자원인 감자에서 과발현 형질전환 식물체를 확보하여 다양한 병 저항성 증가를 통한 작물 생산성 향상에 매우 우수한 기술로 기대되는 바이다.

• 동의생리병리학회지
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• 제19권4호
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• pp.1000-1010
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• 2005

This study was carried out to investigate the comparison of immunomodualtory effects of water-extracted Aconiti lateralis Preparata Radix(PR), Zingiberis Rhizoma(ZR), Cinnamomi Cortex(CC) and Evodiae Fructus(EF). The parameter examined to assess apparent immunomodulatory effect of the water-extracted PR, ZR, CC and EF included the regulation of Nitric oxide (NO). Also, ZR and EF represent the expression of Th1/Th2 type cytokine, the change of B cell phenotype. The water-extracted PR, ZR, CC and EF inhibited NO production and iNOS protein expression in LPS stimulated RAW 264.7 macrophage cells. In the Th1 and Th2 cytokine expression, the water-extracted ZR and EF induced IL-2, IFNr and IL-10 mRNA gene expression. Therefore, it seems that the water-extracted ZR and EF have a inducing effect of Th1 and Th2 type cytokines. In the Flow cytometry analysis, the water-extracted ZR and EF changed B cell phenotype (CD45R/B220), did NOT in PR and CC. The water-extracted PR, ZR, CC and EF have a reducing effect of immune suppression cause by Methotrexate (MTX), an agent of immune suppression. These results suggest that the immunomodulatory effects of the water-extracted ZR and EF may be, in part, associated with the inducing IL-2 and IFNr mRNA gene expression In and regulation of NO production in macrophage cells.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.678-686
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• 1995

SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane of Escherichia coli. In order to study the mechanism of protein secretion in Streptomyces, we have done cloning and sequencing of the Streptomyces coelicolor secY gene by using polymerase chain reaction method. The nucleotide sequence of the gene for SecY from S. coelicolor showed over 58% identity to that of M. luteus. The deduced amino acid sequences were highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments, and especially second, fifth, and tenth segments were particularly conserved, sharing greater than 75% identity with W. lute s SecY. We propose that the conserved membrane-spanning segments actively participate in protein export. In B. subtilis and E. coli, the secY gene is a part of the spc operon, is preceded by the gene coding for ribosomal protein L15, and is likety coupled transcriptionally and translationally to the upstream L15 gene. In the other hand, secY gene of S. coelicolor and M. luteus have its own promoter region, are coupled translationally with adk gene and pr sented in adk operon.

• 대한본초학회지
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• 제30권1호
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• pp.59-65
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• 2015

Objectives : The purpose of this study was to observe the effects of Polygalae Radix(PR) on 4-HNE-induced apoptosis in PC-12 cell. Methods : A MTT assay was conducted to observe the cytotoxicity of Polygalae Radix on the cell viability and the cytoprotective effect of Polygalae Radix against 4-HNE that causes oxidative stress-induced cytotoxicity, and then a western blot was conducted to observe the expression of $TNF-{\alpha}$, caspase-3, Bax and Bcl-2 protein that are important factors involved with apoptosis signaling pathway. Results : The Polygalae Radix water extract $25{\mu}g$, $50{\mu}g$, $100{\mu}g$ and $200{\mu}g/mL$ had no cytotoxicity on the PC-12 cell. The Polygalae Radix water extract $25{\mu}g$, $50{\mu}g$ and $100{\mu}g/mL$ had the cytoprotective effect against 4-HNE that causes cytotoxicity on the PC-12 cell. The Polygalae Radix water extract $50{\mu}g/mL$ significantly suppressed the increase in $TNF-{\alpha}$ protein expression in PC-12 cell. The Polygalae Radix water extract $25{\mu}g$ and $50{\mu}g/mL$ significantly suppressed the increase in caspase-3 protein expression in PC-12 cell. The Polygalae Radix water extract $25{\mu}g$, $50{\mu}g$ and $100{\mu}g/mL$ suppressed the increase in Bax protein expression in PC-12 cell but had no significance. The Polygalae Radix water extract $25{\mu}g$ and $100{\mu}g/mL$ significantly prevented the decrease in Bcl-2 protein expression in PC-12 cell, Conclusions : These results suggest that the Polygalae Radix water extract is effective in inhibiting apoptosis.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.194-203
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• 2009

$HpaG_{Xooc}$, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the $HpaG_{Xooc}$ protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein $HpaG_{Xooc}$ in B. subtilis. The ELISA analysis determined the optimum condition for $HpaG_{Xooc}$ expression in B. subtilis WBHF. The biological function analysis indicated that the protein $HpaG_{Xooc}$ from B. subtilis WBHF elicits hypersensitive response(HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that $HpaG_{Xooc}$ induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

• 미생물학회지
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• 제47권1호
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• pp.22-29
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• 2011

Chromophore로 레티날(vitamin A)을 사용하는 막 단백질인 로돕신은 7개의 막 단백질로 이루어진다. 최근 광화학/생물리학 측정 방법이 다양해지면서, 그에 따른 새로운 특성도 함께 다양하게 보고되고 있다. 하지만, 다양한 측정 환경에 따른 그 광화학/생물리학 특성의 차이점에 대한 비교연구는 없는 상태이다. 첫째, 빛 에너지를 이용하여 수소 이온 펌프 역할 하는 것으로 잘 알려져 있는 roteorhodopsin (PR)은 해양 proteobacteria에서 발견 되었다. 둘째, 한 오페론에서 14 kDa 전달자와 같이 발현되면서 신호 전달 기능을 하는 Anabaena sensory rhodopsin (ASR)이 있다. 이에 본 연구에서는 이 두 미생물 로돕신을 이용하여 다양한 조건에서 그 차이를 살펴보았다. 각 단백질이 막에 끼어 있는 상태(membrane state), polyacrylamide gel에 고정되어 있는 상태, nonionic detergent일종인 DDM (n-dodecyl-${\beta}$-D-maltopyranoside)과, OG (octyl-${\beta}$-D-glucopyranoside)에 녹아 있는 상태, 좀더 자연상태와 유사한 환경을 위해 단백질만 깨끗하게 정제한 다음 다시 Escherichia coli의 지질인 DOPC (1,2-didecanoyl-sn-glycero-3-phosphocholine)에 재조립한 상태, 이렇게 5가지 조건에서 각각의 광화학/생물리학 특징을 흡수스펙트럼(absorption spectrum), 빛-어둠 차이 흡수스펙트럼(light-induced difference spectrum), 포토사이클(photocycle)을 측정하였다. 그 결과 DDM에 녹아 있는 PR과 ASR에서 각 다른 파장에서 M과 O state와 같은 선명한 photointermediate를 가지고, 가장 signal/noise 비율이 좋았다. 본 연구를 통해 막단백질의 다양한 측정 환경에 대한 그 특성의 차이점을 살펴봄으로써 앞으로 다양한 종류의 로돕신 연구에 기초 기반이 될 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제28권6호
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• pp.987-996
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• 2018

Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [$^{35}S$]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE.

• Animal cells and systems
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• 제7권3호
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• pp.213-219
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• 2003

The response of plants to pathogens and wounding is dependent upon very sensitive perception mechanisms. Although genetic approaches have revealed a variety of resistance genes that activate common defense responses, defense-related proteins are not well characterized in plants. Therefore, we used a proteomic approach to determine which defense-related proteins are induced by salicylic acid (SA) and wounding in Chinese cabbage. We found that SA and wounding induce pathogenesis-related protein 1a (PR1a) at both protein and mRNA levels using proteomics and Northern blot analysis, respectively. This indicates that our proteomic approach is useful for identifying defense-related proteins. We also identified several other proteins that are induced by SA or wounding. Among the seven SA-induced proteins identified, four may be defense-related, including defense-related protein, phospholipase D (PLD), resistance protein RPS2 homolog, and L-ascorbate peroxidase. Out of the six wounding-induced proteins identified, three may be defense-related: heat shock cognate protein 70 (HSC70), polygalacturonase, and peroxidase P7. The precise functions of these proteins in plant defense responses await further study. However, identification of the defense-related proteins described in this study should allow us to better understand the mechanisms and signal transduction pathways involved in defense responses in Chinese cabbage.

• Journal of Plant Biotechnology
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• 제36권1호
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• pp.23-29
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• 2009

벼의 저온처리 cDNA 은행에서 분리된 CCCH 형태 zinc finger 단백질인 OsZF2의 기능을 분석하기 위하여 벼에서 CaMV 35S 프로모터 조절하에 OsZF2가 발현(35S:OsZF2)되는 형질전환벼 식물체를 개발하였다. 35S:OsZF2 형질전환벼에 대한 하이그로 마이신저항성 검정을 통해 동형접합체 계통을 선발하고 Northern 발현분석에 의해 OsZF2 유전자가 형질전환체에서 과발현되는 것을 확인하였다. 형질전환체와 대조구인 낙동벼를 100 mM NaCl 첨가 MS 배지에서 키운 후 잎과 뿌리의 길이를 측정하여 내염성 검정을 수행한 결과 대조구에 비해 형질전환체 생육이 다소 양호 한 것으로 나타났다. GMO 포장에서 생육상태를 관찰 한 결과 형질전환체는 생육지연으로 인한 왜화 현상을 나타내며 출수기 또한 열흘 정도 지연되나 등숙기에는 대조구와 같은 초장을 보였다. zinc finger 유전자는 식물체의 발달과 분화 단계 및 환경 스트레스 반응 등 중요한 역할을 하는 것으로 알려져 있으므로 유전체발현 분석으로 하위단계에서 조절되는 유전자 발현 양상을 분석하였다. 35S:OsZF2 전환체에서 낙동벼보다 4배 이상 발현이 증가된 유전자 중에서 게놈 주석에 기초한 기능을 유추하면 신호전달과 관련된 protein kinase, DNA 결합단백질과 대사에 관련된 효소 유전자, 스트레스 반응에 관여하는 일부 유전자 및 병 저항성과 관련된 유전자들의 발현이 증가되었다. 따라서 벼에서 분리된 OsZF2 CCCH type zinc finger 유전자는 벼 성장 발달과 스트레스에 반응하는 상위 조절자로서 기능을 할 것으로 추측된다.

• 대한바이러스학회지
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• 제26권2호
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• pp.191-204
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• 1996

The characterization of the 5 Korean isolates (K96P10, K94P05, K91P55, K87P39, and K82P01) of Japanese encephalitis virus (JEV) was compared with JE virus prototype Nakayama-NIH (NKY-NIH) using prM/M and envelope gene sequences of the JEV genome and phylogenetic analysis. The antigenic analysis of these viruses were done by the cross-hamagglutination inhibition (HI) test using polyclonal antibodies against Korean isolates and NKY-NIH. The sequence homology of the Korean isolates and NKY-NIH ranged between 87.4 % - 95.6 % at the nucleotide level and between 98.2 % - 97.2 % at the amino acid level over the E nucleotides compared. Alignment of E protein amino acid sequences revealed that residue positions E89, E129, E221, E244, E327, E366, E459, and E477 characterized the Korean strains. According to phylogenetic analysis bases on the E nucleotide, there are at least 2 genetic types of JEV existing in Korea and Korean strains were distinct from NKY-NIH. However, the cross HI test results of all the Korean isolates were serologically no different from NKY-NIH strain.