• BMB Reports
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• v.49 no.12
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• pp.693-698
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• 2016

In this study, a tissue-specific GAL4/UAS activation tagging system was used for the characterization of genes which could induce lethality when ubiquitously expressed. A dominant mutant exhibiting stunted growth was isolated and named defective root development 1-D (drd1-D). The T-DNA tag was located within the promoter region of AtTX12, which is predicted to encode a truncated nucleotide-binding leucine-rich repeat (NLR) protein, containing a Toll/interleukin-1 receptor (TIR) domain. The transcript levels of AtTX12 and defense-related genes were elevated in drd1-D, and the misexpression of AtTX12 recapitulated the drd1-D phenotypes. In the presence of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), a key transducer of signals triggered by TIR-type NLRs, a low-level of AtTX12 misexpression induced strong defective phenotypes including seedling lethality whereas, in the absence of EDS1, a high-level of AtTX12 misexpression induced weak growth defects like dwarfism, suggesting that AtTX12 might function mainly in an EDS1-dependent and partially in an EDS1-independent manner.

• Journal of Life Science
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• v.11 no.1
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• pp.30-33
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• 2001

To identify novel components involved in the salt stress signaling pathway of yeast cells, we used mTn3-mediated transposon tagging library and screened mutants displaying enhanced tolerance to NaCl. Southern blot analysis indicated that more than 80% of the sre (salt resistant) mutants possessed only one insertion of the tagged transposon, suggesting that the NaCl resistant phenotype was mediated by a single gene in the majority of the mutants. To define the role of SRE genes in the salt stress signaling pathway, we introduced NaCl stress-inducible ENA1::LacZ construct into the sre mutants and examined the expression of ${\beta}$-galactosidase activity. Interestingly, we could detect high level of ${\beta}$-galactosidase activity without any NaCl treatment in the sre-3, 4, 6 and 7 mutants. These results indicate that SRE-3, 4, and 7 gene are components of salt stress signaling pathway of yeast cells.

• Journal of Plant Biotechnology
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• v.36 no.3
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• pp.294-300
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• 2009

Activation tagging that uses T-DNA vectors containing multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an activation tagged hairy root pool of ginseng (Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the biosynthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quantitative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of triterpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

• Molecules and Cells
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• v.19 no.3
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• pp.342-349
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• 2005

Cytokinins are adenine derivatives that regulate numerous plant growth and developmental processes, including apical and floral meristem development, stem growth, leaf senescence, apical dominance, and stress tolerance. However, not much is known about how cytokinin biosynthesis and metabolism is regulated. We identified a novel Arabidopsis gene, ALL, encoding an aldolase-like enzyme that regulates cytokinin signaling. An Arabidopsis mutant, all-1D, in which ALL is activated by the nearby insertion of the 35S enhancer, exhibited extreme dwarfism with rolled, dark-green leaves and reduced apical dominance, symptomatic of cytokinin-overproducing mutants. Consistent with this, ARR4 and ARR5, two representative primary cytokinin-responsive genes, were significantly induced in all-1D. Whereas SHOOT MERISTEMLESS (STM) and KNAT1, which regulate meristem development, were also greatly induced, expression of REV and PHV that regulate lateral organ polarity was inhibited. ALL encodes an aldolase-like enzyme that belongs to the HpcH/HpaI aldolase family in prokaryotes and is down-regulated by exogenous cytokinin, possibly through a negative feedback pathway. We propose that ALL is involved in cytokinin biosynthesis or metabolism and acts as a positive regulator of cytokinin signaling during shoot apical meristem development and determination of lateral organ polarity.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.10a
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• pp.86-86
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• 2003

• Proceedings of the Korean Society of Crop Science Conference
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• 2003.10a
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• pp.224-225
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• 2003

• Journal of Ginseng Research
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• v.43 no.4
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• pp.645-653
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• 2019

Background: Cytochrome P450 enzymes catalyze a wide range of reactions in plant metabolism. Besides their physiological functions on primary and secondary metabolites, P450s are also involved in herbicide detoxification via hydroxylation or dealkylation. Ginseng as a perennial plant offers more sustainable solutions to herbicide resistance. Methods: Tissue-specific gene expression and differentially modulated transcripts were monitored by quantitative real-time polymerase chain reaction. As a tool to evaluate the function of PgCYP736A12, the 35S promoter was used to overexpress the gene in Arabidopsis. Protein localization was visualized using confocal microscopy by tagging the fluorescent protein. Tolerance to herbicides was analyzed by growing seeds and seedlings on Murashige and Skoog medium containing chlorotoluron. Results: The expression of PgCYP736A12 was three-fold more in leaves compared with other tissues from two-year-old ginseng plants. Transcript levels were similarly upregulated by treatment with abscisic acid, hydrogen peroxide, and NaCl, the highest being with salicylic acid. Jasmonic acid treatment did not alter the mRNA levels of PgCYP736A12. Transgenic lines displayed slightly reduced plant height and were able to tolerate the herbicide chlorotoluron. Reduced stem elongation might be correlated with increased expression of genes involved in bioconversion of gibberellin to inactive forms. PgCYP736A12 protein localized to the cytoplasm and nucleus. Conclusion: PgCYP736A12 does not respond to the well-known secondary metabolite elicitor jasmonic acid, which suggests that it may not function in ginsenoside biosynthesis. Heterologous overexpression of PgCYP736A12 reveals that this gene is actually involved in herbicide metabolism.

• Journal of Korean Society of Forest Science
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• v.87 no.3
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• pp.334-340
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• 1998

This research was conducted to identify plant regulatory regions by gene tagging method. A promoterless GUS coding sequence was introduced to Populus nigra ${\times}$ P. maximowiczii via Agrobacterium strains(LBA4404/EHA101), and putative transgenic poplars were selected by culturing on medium containing G418($60mg/{\ell}$) and by GUS assay. Among them one positive plant was to amplify the native sequences flanking to the introduced GUS gene in plant genome by inverse PCR method and from this 730 by DNA product was obtained. After subcloning and sequencing, it has 88% homology to the Eucalyptus gunnii CAD(cinnamyl alcohol dehydrogenase) gene. The GUS gene fused with the putative promoter reinserted into poplar leaves by particle bombardment method to test the funtional promoter activity. Upon staining with X-gluc, many blue spots appeared on the leaf segments bombarded by the chimeric gene 2-3 days, thus the isolated DNA fragment contain some possible coding region as well as a putative regulatory sequences of poplar CAD gene.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1337-1345
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• 2005

Vibrio vulnificus is the causative agent of foodborne diseases such as gastroenteritis and life-threatening septicemia. Microbial pathogenicity is a complex phenomenon in which expression of numerous virulence factors is frequently controlled by a common regulatory system. In the present study, a mutant exhibiting decreased cytotoxic activity toward intestinal epithelial cells was screened from a library of V. vulnificus mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, an open reading frame, fexA, a homologue of Escherichia coli areA, was identified and cloned. The nucleotide and deduced amino acid sequences of the fexA were analyzed, and the amino acid sequence of FexA from V. vulnificus was $84\%\;to\;97\%$ similar to those of AreA, an aerobic respiration control global regulator, from other Enterobacteriaceae. Functions of the FexA were assessed by the construction of an isogenic mutant, whose fexA gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of fexA resulted in a significant alteration in growth rate under aerobic as well as anaerobic conditions. When compared to the wild-type, the fexA mutant exhibited a substantial decrease in motility and cytotoxicity toward intestinal epithelial cell lines in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the fexA mutant was approximately $10^{1}-10^{2}$ times higher than that of parental wild-type. Therefore, it appears that FexA is a novel global regulator controlling numerous genes and contributing to the pathogenesis as well as growth of V. vulnificus.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.231-236
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• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.