• Journal of Life Science
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• v.28 no.9
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• pp.1030-1041
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• 2018

Abiotic stresses limit the growth and productivity of plants. Cellular adaptation to abiotic stresses requires coordinated regulation in gene expression directed by complex mechanisms. This study used the activation tagging system to identify novel salt stress-responsive genes. The study selected 9 activation tagging lines that showed salt stress-tolerant phenotypes during their germination stages. Thermal asymmetric interlaced-PCR (TAIL-PCR) was used to identify the T-DNA tagging sites on the Arabidopsis genome in selected activation tagging lines, including AT7508, AT7512, AT7527, AT7544, AT7548, and AT7556. RT-PCR analysis showed that ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), and microtubule-associated protein (At5g16730) were activated in AT7508, AT7512, AT7527, AT7544, and AT7556, respectively. Interestingly, in AT7548, both the genes adjacent to the T-DNA insertion site were activated: Arabinogalactan protein 13 (AGP13) (At4g26320) and F-box/RNI-like/FBD-like domains-containing protein (At4g26340). All of the seven genes were newly identified as salt stress-responsive genes from this study. Among them, the expression of ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein gene, and microtubule-associated protein gene were increased under salt-stress condition. In addition, AT7508, AT7527, and AT7544 were more tolerant to salt stress than wild type at seedling development stage, functionally validating the screening results of the activation tagging lines. Taken together, our results demonstrate that the activation tagging system is useful for identifying novel stress-responsive genes.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.307-316
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• 2008

Transposon-mediated insertional mutagenesis is one of powerful strategy for assessing functions of genes in higher plants. In this report, we have selected highly susceptible and tolerance plant by screening about high salt (3% NaCl) and cold stresses ($4^{\circ}C$) from F2 seeds of 30,000 Ac/Ds insertional mutagenesis lines in rice (Oryza sativa L. cv. Dongjin). In order to identify the gene tagging, insertion of Ds element was analyzed by Southern blot and these results revealed that 19 lines were matched genotype of selected lines with phenotype from the first selected 212 lines, and 13 lines have one copy of Ds elements. The Franking Sequence Tags (FSTs) of selected mutant lines showed high similarities with the following known function genes: signal transduction and regulation of gene expression (transpoter, protease family protein and apical meristem family protein), osmotic stress response (heat shock protein, O-methyltransferase, glyceraldehyde-3-phosphate dehydrogenase and drought stress induce protein), vesicle trafficking (SYP 5 family protein) and senescence associated protein. The expression pattern of 19 genes were analyzed using RT-PCR under the abiotic stresses of 9 class; 250mM NaCl, osmotic, drought, 3% $H_2O_2$, $100{\mu}M$ ABA, $100{\mu}M$ IAA, 0.1 ppm 2,4-D, $4^{\circ}C$ cold and $38^{\circ}C$ high temperature. Isolated knock-out genes showed the positive response about 250 mM NaCl, drought, $H_2O_2$, PEG, IAA, 2,4-D, ABA treatment and low ($4^{\circ}C$) and high temperature ($38^{\circ}C$). The results from this study indicate that function of selected knock-out genes could be useful in improving of tolerance to abiotic stresses as an important transcriptional activators in rice.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.289-296
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• 2001

The main objective of this topic is to establish strategies and to plan biotechnology researches which are related to the agricultural improvements especially focusing on the crop breeding in Korea. From 1960's to 1980's government policy had been emphasized to develope high yielding cultivars for the self sufficient supply of the staple food crops. As a result, considerable increase of rice production has been made with accumulating technology and man's powers. Recently genetically modified crops harboring useful characteristics have been developed using biotechnology and released in the developed countries. National research institutes and private companies have been developed biotechnology researches to establish competitive capabilities, however they have not been successfully used in commercialization. Therefore it is necessary to promote the practical. application by connecting molecular technology with conventional breeding. Proposed research projects are; (1) basic researches including plant genome studies, (2) developing new cultivars through gene transformation, (3) screening and producing antioxidants, secondary metabolite substances and edible vaccines. To set a government policy, both domestic and international research trends were reviewed and possibility of success based on the economic view point were discussed. The intellectual property and preservation of environment play a key role to decide the research priority. It is also necessary for us to make one step system for the distribution of research resources such as microorganisms, genes cloned, plant seeds and research informations for promoting research activities.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.88-89
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• 2013

A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.

• Korean Journal of Environmental Biology
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• v.34 no.4
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• pp.304-313
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• 2016

One of the issues currently facing nuclear power plants is how to store spent nuclear waste materials which are contaminated with radionuclides such as $^{134}Cs$, $^{135}Cs$, and $^{137}Cs$. Bioremediation processes may offer a potent method of cleaning up radioactive cesium. However, there have only been limited reports on $Cs^+$ tolerant bacteria. In this study, we report the isolation and identification of $Cs^+$ tolerant bacteria in environmental soil and sediment. The resistant $Cs^+$ isolates were screened from enrichment cultures in R2A medium supplemented with 100 mM CsCl for 72 h, followed by microbial community analysis based on sequencing analysis from 16S rRNA gene clone libraries(NCBI's BlastN). The dominant Bacillus anthracis Roh-1 and B. cereus Roh-2 were successfully isolated from the cesium enrichment culture. Importantly, B. cereus Roh-2 is resistant to 30% more $Cs^+$ than is B. anthracis Roh-1 when treated with 50 mM CsCl. Growth experiments clearly demonstrated that the isolate had a higher tolerance to $Cs^+$. In addition, we investigated the adsorption of $0.2mg\;L^{-1}$ $Cs^+$ using B. anthracis Roh-1. The maximum $Cs^+$ biosorption capacity of B. anthracis Roh-1 was $2.01mg\;g^{-1}$ at pH 10. Thus, we show that $Cs^+$ tolerant bacterial isolates could be used for bioremediation of contaminated environments.

• Radiation Oncology Journal
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• v.19 no.4
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• pp.389-396
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• 2001

Purpose : To detect differentially expressed genes in the patients with uterine cervical cancer during the radiation therapy. Materials and Methods : In patients with biopsy proven uterine cervical cancer, we took tumor tissue just before radiation therapy and at 40 minutes after external irradiation of 1.8 Gy. Total RNAs isolated from non-irradiated and irradiated tumor tissue samples were analyzed using the differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs(mRNAs) were eluted, and cloned. The differential expression of the corresponding mRNAs was confirmed by reverse northern blot. Differentially expressed cDNA bands were sequenced. Nucleotide sequence data were analyzed in the Gene Bank and EMBL databases via the BLAST network sewer to identify homologies to known genes or cDNA fragments. Expression pattern of down-regulated clone was examined using RT-PCR in S patients undergoing radiotherapy. Results : We identified 18 differentially expressed bands by DDRT-PCR, which were eluted and cloned. There were 10 up-regulated clones and 1 down-regulated clone in reverse northern blot. One cDNA fragment had homology to chemokine receptor CXCR4, four were identified as Human ESTs in the EMBL database in EST clones. Down-regulated CxCa-11 was also down regulated in all patients. Conclusion : Using the DDRT-PCR, we have identified 10 up-regulated and 1 down-regulated clone(s) in the patients with uterine cervical cancer during the radiation therapy. The clinical relevance and the functions of these genes will be further investigated.

• Journal of Life Science
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• v.14 no.4
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• pp.650-656
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• 2004

Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.

• Journal of Dairy Science and Biotechnology
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• v.34 no.4
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• pp.203-216
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• 2016

Probiotic microorganisms are thought to provide health benefits when consumed. In 2001, the World Health Organization defined probiotics as "live microorganisms which confer a health benefit on the host, when administered in adequate amounts." Three methods for screening potential probiotics have currently widely available. (1) In vitro assays of potential probiotics are preferred because of their simplicity and low cost. (2) The use of in vivo approaches for exploring various potential probiotics reflects the enormous diversity in biological models with various complex mechanisms. (3) Potential probiotics have been analyzed using several genetic and omics technologies to identify gene expression or protein production patterns under various conditions. However, there is no ideal procedure for selecting potential probiotics than testing cadidate strains on the target population. Hence, in this review, we provide an overview of the different methodologies used to identify new probiotic strains. Furthermore, we describe futre perspectives for the use of in vitro, in vivo and omics in probiotic research.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.64 no.4
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• pp.422-431
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• 2019

Severe droughts in spring have occurred frequently in Korea in recent years, exerting a critical impact on corn yield. Therefore, it is necessary to find physiological and/or molecular indicators of the response to drought stress in maize plants. In this study, we investigated the effects of water-deficit stress on two Korean elite F₁ maize hybrids, Ilmichal and Gwangpyeongok, by withholding water for 10 days at tassel initiation. The water deficit drastically reduced the relative leaf water content, leaf number, leaf area, and stem length, leading to dry matter reduction. Moreover, it reduced the SPAD values and stomatal conductance of leaves in drought-stressed plants of both hybrids. Importantly, the number of leaves and SPAD value were non-destructive and easy to investigate in response to water-deficit stress, suggesting that they may be useful indicators for screening drought-tolerant genetic resources. We detected more than 100 spots that were differentially accumulated under drought stress. Of these spots, a total of 21 protein spots (≥1.5-fold) from drought-exposed maize leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Functional annotation using Gene Ontology analysis revealed that most of the identified proteins were involved in carbohydrate metabolism, stress response fatty acid catabolism, photosynthesis, energy metabolism, and transport. The protein expression levels were increased in both Ilmichal and Gwangpyeongok, except for triosephosphate isomerase, fructose-bisphosphate aldolase, and an uncharacterized protein. The lactoylglutathione lyase delta (3,5)-delta (2,4)-dienoyl-CoA isomerase was overexpressed in Gwangpyeongok only. The results obtained from this study suggest that the drought-specific genes may be useful as molecular markers for screening drought-tolerant maize genotypes.

• Journal of Plant Biotechnology
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• v.5 no.3
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• pp.143-148
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.