• Molecules and Cells
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• 제40권8호
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• pp.577-586
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• 2017

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana, which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the ${\beta}-glucuronidase$ reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis, which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout (cys5) plants grown under HS conditions. The HS tolerance of AtCYS5-overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5. Although no HS elements were identified in the 5'-flanking region of AtCYS5, canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABAtreated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.263-268
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• 2005

Porcine epidemic diarrhea virus (PEDV)는 돼지의 급성장염을 유발하여 설사 증상을 일으키는 바이러스이다. 본 연구에서는 고구마 저장뿌리 고발현 sporamin 프로모터 및 CaMV 35S promoter를 이용하여 PEDV 항원단백질을 생산하는 고구마 식물체를 개발하고자 하였다. 형질전환 벡터를 제작하기 위하여 PEDV에서 항원성이 알려진 스파이크 단백질의 일부분을 암호화하는 유전자를 PCR로 합성하였다. 고구마 [Ipomoea batatas (L.) Lam] 율미 품종의 배발생 캘러스를 재료로 하여 Agrobacterium tumefaciens을 매개로 형질전환하였다. 선발배지 (MS medium, 1 mg/L 2,4-D, 100 mg/L kanamycin, and 400 mg/L claforan)에서 배발생 캘러스를 3주 간격으로 4개월 동안 계대배양하여 카나마이신 저항성 캘러스를 선발하였다. 선발된 배발생 캘러스를 호르몬을 제거한 배지로 옮겨 체세포배를 유도하였으며 이후 shoot과 뿌리가 형성되었다. 재분화된 소식물체를 대상으로 PCR 분석으로 카나마이신 저항성 재분화 개체의 50% 이상이 도입 유전자를 가지고 있었으며 이들 식물체를 대상으로 Southern blot 분석하여 PEDV 유전자가 고구마 식물체의 게놈으로 안정적으로 도입되었음을 확인하였다. 형질전환 식물체에서 도입유전자의 발현을 RT-PCR로 분석한 결과 PEDV의 spike 유전자가 높은 수준으로 발현하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.55-60
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• 1998

Various Saccharomyces cerevisiae strains were transformed with a 2 ${\mu}$-based multicopy expression plasmid, pYIGP, carrying Kluyveromyces marxianus inulinase gene under the control of GAPDH promoter. Among then two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinant S. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.

• The Korean Journal of Physiology and Pharmacology
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• 제18권5호
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• pp.403-409
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• 2014

The Bis protein is known to be involved in a variety of cellular processes including apoptosis, migration, autophagy as well as protein quality control. Bis expression is induced in response to a number of types of stress, such as heat shock or a proteasome inhibitor via the activation of heat shock factor (HSF)1. We report herein that Bis expression is increased at the transcriptional level in HK-2 kidney tubular cells and A172 glioma cells by exposure to oxidative stress such as $H_2O_2$ treatment and oxygen-glucose deprivation, respectively. The pretreatment of HK-2 cells with N-acetyl cysteine, suppressed Bis induction. Furthermore, HSF1 silencing attenuated Bis expression that was induced by $H_2O_2$, accompanied by increase in reactive oxygen species (ROS) accumulation. Using a series of deletion constructs of the bis gene promoter, two putative heat shock elements located in the proximal region of the bis gene promoter were found to be essential for the constitutive expression is as well as the inducible expression of Bis. Taken together, our results indicate that oxidative stress induces Bis expression at the transcriptional levels via activation of HSF1, which might confer an expansion of antioxidant capacity against pro-oxidant milieu. However, the possible role of the other cis-element in the induction of Bis remains to be determined.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.267-276
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• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• BMB Reports
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• 제47권1호
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• pp.27-32
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• 2014

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots.

• Molecules and Cells
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• 제27권2호
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• pp.217-223
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• 2009

Development of symbiotic root nodules in legumes involves the induction and repression of numerous genes in conjunction with changes in the level of phytohormones. We have isolated several genes that exhibit differential expression patterns during the development of soybean nodules. One of such genes, which were repressed in mature nodules, was identified as a putative aldo/keto reductase and thus named Glycine max aldo/keto reductase 1 (GmAKR1). GmAKR1 appears to be a close relative of a yeast aldo/keto reductase YakC whose in vivo substrate has not been identified yet. The expression of GmAKR1 in soybean showed a root-specific expression pattern and inducibility by a synthetic auxin analogue 2,4-D, which appeared to be corroborated by presence of the root-specific element and the stress-response element in the promoter region. In addition, constitutive overexpression of GmAKR1 in transgenic soybean hairy roots inhibited nodule development, which suggests that it plays a negative role in the regulation of nodule development. One of the Arabidopsis orthologues of GmAKR1 is the ARF-GAP domain 2 protein, which is a potential negative regulator of vesicle trafficking; therefore GmAKR1 may have a similar function in the roots and nodules of legume plants.

• BMB Reports
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• 제32권4호
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• pp.399-404
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• 1999

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), which catalyzes the ring meta-cleavage of 2,3-dihydroxybiphenyl, is encoded by the phnQ gene of biphenyl- and phenanthrene-degrading Pseudomonas sp. strain DJ77. We determined the nucleotide sequence of a DNA fragment of 1497 base pairs which included the phnQ gene. The fragment lncluded an open reading frame of 903 base pairs to accommodate the enzyme. The predicted amino acid sequence of the enzyme subunit consisted of 300 residues. In front of the gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli. The deduced amino acid sequence of the PhnQ enzyme exhibited 85.6% identity with that of the corresponding enzyme in Sphingomonas yanoikuyae Q1 (formerly S. paucimobilis Q1) and 22.1% identity with that of catechol 1,2,3-dioxygenase from the same DJ77 strain. PhnQ showed broader substrate preference than previously-cloned PhnE, catechol 2,3-dioxygenase. Ten amino acid residues, considered to be important for the role of extradiol dioxygenases, were conserved.

• BMB Reports
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• 제41권3호
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• pp.204-209
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• 2008

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phyto-planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1127-1134
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• 2009

Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.