• Title/Summary/Keyword: High-level expression

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Development of high tryptophan GM rice and its transcriptome analysis (고 함량 트립토판 생산 GM 벼 개발 및 전사체 분석)

  • Jung, Yu Jin;Nogoy, Franz Marielle;Cho, Yong-Gu;Kang, Kwon Kyoo
    • Journal of Plant Biotechnology
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    • v.42 no.3
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    • pp.186-195
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    • 2015
  • Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive ${\alpha}$ subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

Characterization of Ovarian Cytochrome $P450_{C17}$ (17 ${\alpha}-hydroxylase$/17,20-lyase) in Rana dybowski (북방산 개구리 난소의 Cytochrome $P450_{C17}$ 유전자 특성)

  • Kang, Hae-Mook
    • Development and Reproduction
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    • v.10 no.2
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    • pp.127-133
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    • 2006
  • [ $17\;{\alpha}-hydroxylase/17,20-lyase(P450_{C17})$ ] is the key enzyme mediating the conversion of progesterone to $17\;{\alpha}-hydroxyprogesterone$, ultimately to androstenedione during steroidogenesis. R. dybowskii's ovarian $P450_{C17}$ cDNA was cloned to understand the regulatory mechanism of ovarian steroidogenic pathway at the molecular level in amphibian. A 2.5kb cDNA clone encoding a single open-reading frame with a 519 deduced amino acid was isolated with the screening of ovarian cDNA library. This sequence contained the three highly conserved domains as seen in $P450_{C17}$ of other species. The comparison of amino acid sequence of Rana $P450_{C17}$ with other animal's $P450_{C17}$ showed relatively high identity with 76% in Xenopus, 63% in chicken, 60% in rainbow trout, and 45% in human. Phylogenic analysis also indicated that Rana $P450_{C17}$ gene was evolutionary well conserved among vertebrate. Northern analysis indicated that the two different sizes of $P450_{C17}$ transcripts with approximately 2.5 and 3.6kb were detected in ovary tissue, but not in other tissues. The expression vector of Rana $P450_{C17}$ clearly showed the $17\;{\alpha}-hydroxylase$ activity converting the exogenous progesterone into $17\;{\alpha}-hydroxyprogesterone$ in the nonsteroidogenic COS-1 cells. Therefore, Rana $P450_{C17}$ cDNA is very useful to investigate the molecular mechanism of the ovarian steroidogenesis in amphibian.

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Proteome analysis of sorghum leaf and root in response to heavy metal stress

  • Roy, Swapan Kumar;Cho, Seong-Woo;Kwon, Soo Jeong;Kamal, Abu Hena Mostafa;Lee, Dong-Gi;Sarker, Kabita;Lee, Moon-Soon;Xin, Zhanguo;Woo, Sun-Hee
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2017.06a
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    • pp.24-24
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    • 2017
  • Heavy metals at toxic levels have the capability to interact with several vital cellular biomolecules such as nuclear proteins and DNA, leading to oxidative stress in plants. The present study was performed to explore the metal tolerance mechanism in Sorghum seedling. Morpho-physiological and metal ions uptake changes were observed prominently in the seedlings when the plants were subjected to different concentrations of $CuSO_4$ and $CdCl_2$. The observed morphological changes revealed that the plants treated with Cu and Cd displayed dramatically altered shoot lengths, fresh weights, and relative water content. In addition, the concentration of Cu and Cd was markedly increased by treatment with Cu and Cd, and the amount of interacting ions taken up by the shoots and roots was significantly and directly correlated with the applied level of Cu and Cd. Using the 2-DE method, a total of 24 and 21 differentially expressed protein spots from sorghum leaves and roots respectively, 33 protein spots from sorghum leaves under Cd stress were analyzed using MALDI-TOF/TOF MS. However, the over-expression of GAPDH plays a significant role in assisting Sorghum bicolor to attenuate the adverse effects of oxidative stress caused by Cu, and the proteins involved in resistance to stress helped the sorghum plants to tolerate high levels of Cu. Significant changes were absorbed in the levels of proteins known to be involved in carbohydrate metabolism, transcriptional regulation, translation and stress responses. In addition, the up-regulation of glutathione S-transferase and cytochrome P450 may play a significant role in Cd-related toxicity and stress responses. The results obtained from the present study may provide insights into the tolerance mechanism of seedling leaves and roots in Sorghum under heavy metal stress.

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Power affects emotional awareness: The moderating role of emotional intelligence and goal-relevance (정서인식과 권력의 관계: 정서지능과 목표관련성의 조절효과 검증)

  • Lee, Suran;Lee, Won Pyo;Kim, Kaeun;Youm, Joon-Kyoo;Sohn, Young Woo
    • Science of Emotion and Sensibility
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    • v.16 no.4
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    • pp.433-444
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    • 2013
  • The purpose of this study is to investigate the moderating role of emotional intelligence (EI) and goal-relevance in the relationship between power and emotional awareness. In Study 1, participants were ask to correctly indicate presented facial expressions of others after completing EI survey. Half of the participants were randomly assigned to the "power" condition and the other half to the "powerless" condition. In Study 2, goal-relevance of expressed emotion was manipulated. The results showed that EI moderated the relationship between power and emotion decoding ability. While participants with high and low levels of EI were not significantly affected by power condition, participants with middle level of EI were strongly influenced by the effect of power. In addition, the role of goal-relevance significantly moderated the relationship between power and emotional awareness. When correctly indicating other's emotion became important and thus emotional awareness was strongly associated with participants' goal, those who had power performed better than before.

SNR Estimation Based on Correlation of Decision Feedback Signal in OFDM System (OFDM 시스템에서 Decision Feedback 신호의 상관 관계를 이용하는 SNR 추정)

  • Kim, Seon-Ae;Ryu, Heung-Gyoon;Lee, Seung-Jun;Ko, Dong-Kuk
    • The Journal of Korean Institute of Electromagnetic Engineering and Science
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    • v.21 no.9
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    • pp.995-1004
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    • 2010
  • In the channel-varying environment, it is very important to estimate the signal to noise ratio(SNR) of received signal and to transmit the signal effectively for the modern communication system. The performance of existing non-data-aided (NDA) SNR estimation methods are substantially degraded for high level modulation scheme such as M-ary APSK or QAM. In this paper, we propose a SNR estimation method which uses zero point auto-correlation of received signal per block and auto-/cross- correlation of decision feedback signal in OFDM system. Proposed method can be studied into two Types; Type 1 can estimate SNR by zero point auto-correlation of decision feedback signal based on the second moment property. Type 2 uses both zero point auto-correlation and cross-correlation based on the fourth moment property. In block-by-block reception of OFDM system, these two SNR estimation methods can be possible for the practical implementation due to correlation based the estimation method and they show more stable estimation performance than the previous SNR estimation methods. Also, we mathematically derive the SNR estimation expression according to computational difference of auto-/cross-correlation. Finally, Monte Carlo simulations are used to verify the proposed method.

Impact on Inflammation and Recovery of Skin Barrier by Nordihydroguaiaretic Acid as a Protease-Activated Receptor 2 Antagonist

  • Kim, Hyo-Young;Goo, Jung-Hyun;Joo, Yeon-Ah;Lee, Ha-Yoen;Lee, Se-Mi;Oh, Chang-Taek;Ahn, Soo-Mi;Kim, Nam-Hoon;Hwang, Jae-Sung
    • Biomolecules & Therapeutics
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    • v.20 no.5
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    • pp.463-469
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    • 2012
  • Atopic dermatitis is a chronic, inflammatory disease of the skin with increased transepidermal water loss. Both an abnormal inflammatory response and a defective skin barrier are known to be involved in the pathogenesis of atopic dermatitis. Protease activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is activated by both trypsin and a specific agonist peptide, SLIGKV-$NH_2$. PAR2 is expressed in suprabasal layers of the epidermis and regulates inflammatory responses and barrier homeostasis. In this study, we show that nordihydroguaiaretic acid (NDGA) inhibits the PAR2-mediated signal pathway and plays a role in skin barrier recovery in atopic dermatitis. Specifically, NDGA reduces the mobilization of intracellular $Ca^{2+}$ in HaCaT keratinocytes by down-regulating inflammatory mediators, such as interleukin-8, thymus and activation-regulated chemokine and intercellular cell adhesion molecule-1 in HaCaT keratinocytes. Also, NDGA decreases the protein expression of involucrin, a differentiation maker of keratinocyte, in both HaCaT keratinocytes and normal human epidermal keratinocytes. We examined NDGA-recovered skin barrier in atopic dermatitis by using an oxazolone-induced atopic dermatitis model in hairless mice. Topical application of NDGA produced an increase in transepidermal water loss recovery and a decrease in serum IgE level, without weight loss. Accordingly, we suggest that NDGA acts as a PAR2 antagonist and may be a possible therapeutic agent for atopic dermatitis.

Induction of DNA Damage in L5178Y Cells Treated with Gold Nanoparticle

  • Kang, Jin-Seok;Yum, Young-Na;Kim, Joo-Hwan;Song, Hyun-A;Jeong, Jin-Young;Lim, Yong-Taik;Chung, Bong-Hyun;Park, Sue-Nie
    • Biomolecules & Therapeutics
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    • v.17 no.1
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    • pp.92-97
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    • 2009
  • As nanomaterials might enter into cells and have high reactivity with intracellular structures, it is necessary to assay possible genotoxic risk of them. One of these approaches, we investigated possible genotoxic potential of gold nanoparticle (AuNP) using L5178Y cells. Four different sizes of AuNP (4, 15, 100 or 200 nm) were synthesized and the sizes and structures of AuNP were analyzed using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and stability was analyzed by a UV/Vis. Spectrophotometer. Cytotoxicity was assessed by direct cell counting, and cellular location was detected by dark field microscope at 6, 24 and 48 h after treatment of AuNP. Comet assay was conducted to examine DNA damage and tumor necrosis factor (TNF)-${\alpha}$ mRNA level was assay by real-time reverse transcription polymerase chain reaction. Synthetic AuNP (4, 50, 100 and 200 nm size) had constant characteristics and stability confirmed by TEM, SEM and spectrophotometer for 10 days, respectively. Dark field microscope revealed the location of AuNP in the cytoplasm at 6, 24 and 48 h. Treatment of 4 nm AuNP induced dose and time dependent cytotoxicity, while other sizes of AuNP did not. However, Comet assay represented that treatment of 100 nm and 200 nm AuNP significantly increased DNA damage compared to vehicle control (p <0.01). Treatment of 100 nm and 200 nm AuNP significantly increased TNF-${\alpha}$ mRNA expression compared to vehicle control (p<0.05, p<0.01, respectively). Taken together, AuNP induced DNA damage in L5178Y cell, associated with induction of oxidative stress.

Tolerance to Potato Soft Rot Disease in Transgenic Potato Expressing Soybean Ferritin Gene (대두 철분결합단백질 유전자 발현 형질전환 감자의 감자무름병 방어 증진효과)

  • Bae, Shin-Chul;Yeo, Yun-Soo;Heu, Sung-Gi;Hwang, Duk-Ju;Byun, Myung-Ok;Go, Seung-Joo
    • Journal of Plant Biotechnology
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    • v.29 no.4
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    • pp.229-233
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    • 2002
  • Ferritin is ubiquitous in bacteria, animals and plants. Ferritin is thought to play two main roles in living cells to provide iron for the synthesis of iron protein such as ferretoxin and cytochromes and to prevent damage from radicals produced by iron/dioxygen interaction. To enhance the resistance of potato to Erwinia carotovora, the soybean ferritin gene was introduced into the potato either under CaMV 35S or hsr203J promoter. Potato transgenic plants were screened by PCR analysis using specific primers to the ferritin gene. Expression of ferritin gene under CaMV 35S and hsr203J promoter in potato transgenic plants was confirmed by northern blot analysis. hsr203J promoter known to pathogen inducible in tobacco drives the induction upon Phytophthora infestan in potato and the transcript level of ferritin gene was extremely high after 24 hours post inoculation. One of transformants under CaMV 35S promoter was increased 2.5 fold than untransformant. Each one of transgenic potato containing gene promoter CaMV 35S and hsr203J-ferrtin fusion exhibited tolerance against potato soft rot.

Isolation of cDNA Encoding Low Temperature-inducible L-asparaginase from Soybean (Glycin max) (저온 스트레스에 발현이 유도되는 콩의 L-asparaginase 유전자의 분리)

  • Park, Seong-Whan;Kim, Kee-Young;Chen, Liang;Lee, Jai-Heon
    • Journal of Plant Biotechnology
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    • v.29 no.2
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    • pp.99-104
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    • 2002
  • Suppression subtractive hybridization (SSH) was used to isolate wound-induced cDNAs from wounded soybean. One of low-temperature-inducible cDNA, slti182 showed high homology with genes encoding 1-asparaginase. The full length cDNA of slti182, deginated GmASP1, is 1258 bp long and contains an open reading frame consisted of 326 amino acids. CmASP1 protein showed the highest identity (84%) with putative asparaginase from A. thaliana (AB012247), but it showed only 55% identity with another isoform of A. tathaliana (Z34884). The expression of GmASP1 during low temperature stress started to increase 3 hours after treatment, reached the maximum at 6 hour, and then decreased to the initial level at 48 hours. The amount of GmASP1 transcripts increased again when low-temperature-treated plants were transferred to room temperature, The present study suggests that GmASP1 may function to accelerate the protein synthesis which is important in the early response to low temperature.

Quality characteristics of buckwheat Soksungjang manufactured by Bacillus subtilis HJ18-4 (Bacillus subtilis HJ18-4를 이용하여 제조한 메밀 속성장의 품질특성)

  • Park, Na Young;Lee, Sun Young;Kim, Ji Yeun;Choi, Hye Sun
    • Food Science and Preservation
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    • v.20 no.5
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    • pp.699-704
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    • 2013
  • Buckwheat Soksungjang (BS) is a bealmijang manufactured with buckwheat and soybeans. We manufactured BS using Bacillus subtilis HJ18-4 (HJ18-4), which has high enzyme activities and antibacterial effects. HJ18-4 was inoculated in a different process during the BS manufacturing, which was the meju-making time (Treat 1), and the salt water time was added (Treat 2). The physiochemical and microbial characteristics of the BS were analyzed. As a result, the total aerobic counts (7~8 log CFU/mL) in the BS increased after 15 days of fermentation. Especially, Treat 1 showed higher total aerobic counts and amino-type nitrogen (65.38~202.52 mg%) than Treat 2. During the BS fermentation, the reduction of the sugar contents and the enzyme (protease and amylase) activities decreased. In the relative quantitative expression level of PlcR, Treat 1 did not show toxin gene expressions at the end of the fermentation on Day 23. Treat 1 showed suitable B. cereus physiochemical quality characteristics and inhibition effects. When the modified-form type of fermented soybean paste was manufactured with a single starter, it could not reproduce the natural fermentation quality. These results suggest that the addition of a starter (HJ18-4) in the Meju manufacturing process could enhance the quality characteristics of the manufactured BS via natural fermentation and by suppressing B. cereus.