• Title/Summary/Keyword: reverse-transcription-PCR

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Transcriptional activation of anthocyanin structural genes in torenia cv. Kauai rose by overexpression of anthocyanin regulatory transcription factors

  • Xu, Jun-Ping;Naing, Aung Htay;Kim, Chang-Kil
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2018.10a
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    • pp.33-33
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    • 2018
  • This study was conducted to examine the role of the transcription factors (TFs) (RsMYB1 and mPAP1+B-Peru) in the regulation of anthocyanin biosynthesis in the ornamental torenia cv. Kauai rose. In this study, we could produce several putative transgenic lines overexpressing the TFs via Agrobacterium-mediated transformation, and presence of the TFs in the randomly selected five transgenic lines was confirmed using polymerase chain reaction (PCR). According to results of reverse transcription-PCR analysis (RT-PCR), the expression of the TFs in all transgenic lines and of the anthocyanin structural genes (CHS, F3H, DFR, and ANS) in all transgenic lines and WT plants were distinctly detectable. However, transcript levels of the structural genes expressed in the transgenic lines overexpressing TFs were significantly higher than those expressed in WT plants. Therefore, it is suggested that anthocyanin content in flowers of the transgenic torenia would be significantly higher than that in flowers of WT plants. Moreover, these results indicate that the TFs (RsMYB1 and mPAP1+B-Peru) could be exploited as potential anthocyanin regulatory TFs to enhance anthocyanin content in the other horticultural plants.

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Identification and Differentiation of Cucumber Mosaic Virus Isolated from Forsythia koreana (CMV-Fk) Using PCR Techniques (PCR기법을 이용한 오이 모자이크 바이러스 개나리 분리주(CMV-Fk)의 동정과 구분)

  • 이상용;박선정;최장경
    • Korean Journal Plant Pathology
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    • v.14 no.4
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    • pp.308-313
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    • 1998
  • Reverse transcription and polymerase chain reaction (RT-PCR) techiniques were used to identification and differentiation of cucumber mosaic virus isolated from Forsythia koreana (CMV-Fk). RT-PCT used by two set of 20-mer primers one was CMV-common primers and another was CMV subgroup I-specific primers designed in a conserved region of the 3' end of CMV RNA3, amplified about 490 bp and 200 bp DNA fragments from CMV-Fk, respectively. CMV could be detected by RT-PCR at a dilution as low as 10-4 in forsythia crude sap extracts. Restriction enzyme analysis of RT-PCR products using EcoRI and MspI showed that CMV-Fk belonged to CMV subgroup I. But, analysis of RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) showed heterogeneity of RNA3 between CMV-Fk and CMV-Y as a member of subgroup I.

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Citrus unshiu Water Extract Inhibits Trypsin-induced $TNF-{\alpha}$ and Tryptase Productions by Blocking the ERK Phosphorylation and Trypsin Activity

  • Kang, Ok-Hwa;Kim, Dae-Ki;Lee, Young-Mi
    • Natural Product Sciences
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    • v.10 no.5
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    • pp.211-216
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    • 2004
  • Citrus unshiu (Rutaceae) has long been known as an anti-inflammatory and anti-allergic agent. In the present study, the inhibitory effect of CUWE (Citus unshiu water extract) on the production of $TNF-{\alpha}$ and tryptase was examined. In addition, a possible mechanism for the inhibition of trypsin-stimulated human leukemic mast cell-1 (HMC- 1 ) activation was determined. To do so, $TNF-{\alpha}$ production from the HMC-1 cells that were stimulated by trypsin (100 nM) in the presence or absence of CUWE $(10,\;100,\;and\;100\;{\mu}g/ml)$ was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. The tryptase production was evaluated by reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. Results showed that the CUWE inhibited production of both $TNF-{\alpha}$ and tryptase from the trypsin-stimulated HMC-1 in a dose-dependent manner. The CUWE a1so inhibited the ERK phosphorylation and trysin activity. These results indicate that the CUWE had an inhibitory effect on $TNF-{\alpha}$ and the tryptase productions by blocking the ERK phosphorylation and trypsin activity.

Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

  • Kwon, Ji Yeon;Ryu, Ki Hyun;Choi, Sun Hee
    • The Plant Pathology Journal
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    • v.29 no.3
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    • pp.338-343
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    • 2013
  • A detection system based on a multiplex reverse transcription (RT) polymerase chain reaction (PCR) was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV), lily mottle virus (LMoV), lily symptomless virus (LSV). Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single- or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV) by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

Mcl-PHAs Produced by Pseudomonas sp. Gl01 Using Fed-Batch Cultivation with Waste Rapeseed Oil as Carbon Source

  • Mozejko, Justyna;Wilke, Andreas;Przybylek, Grzegorz;Ciesielski, Slawomir
    • Journal of Microbiology and Biotechnology
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    • v.22 no.3
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    • pp.371-377
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    • 2012
  • The present study describes medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production by the Pseudomonas Gl01 strain isolated from mixed microbial communities utilized for PHAs synthesis. A two-step fed-batch fermentation was conducted with glucose and waste rapeseed oil as the main carbon source for obtaining cell growth and mcl-PHAs accumulation, respectively. The results show that the Pseudomonas Gl01 strain is capable of growing and accumulating mcl-PHAs using a waste oily carbon source. The biomass value reached 3.0 g/l of CDW with 20% of PHAs content within 48 h of cultivation. The polymer was purified from lyophilized cells and analyzed by gas chromatography (GC). The results revealed that the monomeric composition of the obtained polyesters depended on the available substrate. When glucose was used in the growth phase, 3-hydroxyundecanoate and 3-hydroxydodecanoate were found in the polymer composition, whereas in the PHAs-accumulating stage, the Pseudomonas Gl01 strain synthesized mcl-PHAs consisting mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate. The transcriptional analysis using reverse-transcription real-time PCR reaction revealed that the phaC1 gene could be transcribed simultaneously to the phaZ gene.

Detection of RNA Mycoviruses in Wild Strains of Lentinula edodes in Korea

  • Kim, Eunjin;Park, Mi-Jeong;Jang, Yeongseon;Ryoo, Rhim;Ka, Kang-Hyeon
    • The Korean Journal of Mycology
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    • v.49 no.3
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    • pp.285-294
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    • 2021
  • In general, mycoviruses remain latent and rarely cause visible symptoms in fungal hosts; however, some viral infections have demonstrated abnormal mycelial growth and fruiting body development in commercial macrofungi, including Lentinula edodes. Compared to other cultivated mushrooms, L. edodes is more vulnerable to viral infections as it is still widely cultivated under near-natural conditions. In this study, we investigated whether Korean wild strains of L. edodes were infected by RNA mycoviruses that have previously been reported in other parts of the world (LeSV, LePV1, LeV-HKB, LeNSRV1, and LeNSRV2). Using specific primer sets that target the RNA-dependent RNA polymerase genes of each of the RNA mycovirus, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral infection. Viral infection was detected in about 90% of the 112 wild strains that were collected in Korea between 1983 and 2020. Moreover, multiple infections with RNA mycoviruses were detected in strains that had normal fruiting bodies. This work contributes to our understanding of the distribution of RNA mycoviruses in Korea and the impact of multiple viral infections in a single strain of L. edodes.

Effect of pH on the expression of RsMYB1 that regulates anthocyanin production in Petunia plants

  • Lee, Deuk Bum;Ai, Trinh Ngoc;Naing, Aung Htay;Kim, Chang Kil
    • Journal of Plant Biotechnology
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    • v.45 no.1
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    • pp.30-35
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    • 2018
  • We established an in vitro system to investigate transcription levels of the RsMYB1 gene expressed in T2 20-day-old transgenic Petunia plants (three independent lines: PhRs1, PhRs2, and PhRs3), and the association between those transcription levels and anthocyanin production at various pH values (3.0 to 8.0) for a period of 10 days. All the lines treated with pH 5.0-7.0 exhibited increased anthocyanin content and delays in growth compared to the wild-type (WT) seedlings. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that the enhancement of anthocyanin production in the transgenic lines was due to the upregulation of RsMYB1 transcription at various pH values. The results suggest that pH value can control expression of RsMYB1 which is associated with anthocyanin production.

Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children (소아에서 폐렴구균 집락률 측정을 위해 비인두 흡인 물의 총 RNA를 이용한 실시간 중합효소 연쇄반응법)

  • Kim, Young Kwang;Lee, Kyoung Hoon;Yun, Ki Wook;Lee, Mi Kyung;Lim, In Seok
    • Pediatric Infection and Vaccine
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    • v.23 no.3
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    • pp.194-201
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    • 2016
  • Purpose: Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae. Methods: In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, real-time PCR, and real-time RT-PCR. Results: The concordance rate between culture findings and the results of real-time PCR was 0.922 (P<0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia -positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae -positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively. Conclusions: This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

Analysis and Detection of Coast Protein Gene of Barley Yellow Mosaic Virus and Barley Mield Mosaic Virus by RT-PCR (RP-PCR을 이용한 보리누른모자이크바이러스 (BaYMV)와 보리마일드모자이크바이러스(BaMMV)의 외피단백질 유전자 검정 및 해석)

  • 이귀재
    • Korean Journal Plant Pathology
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    • v.14 no.4
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    • pp.314-318
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    • 1998
  • Using the reverse transcription polymerase chain reaction (RT-PCR), a rapid and sensitive assay method for the detection and identification of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) was adapted. Two units of primers from each virus were selected and used for the determination of two different viruses. PCR fragments of BaYMV (ca. 0.9kb) and BaMMV (ca. 0.8kb) were obtained from the designed method for the assay of BaYMV and BaMMV coat protein. PT-PCR fragments were cloned using vector pT7 Blue and the sequences of the selected clones were analyzed. coat protein of BaYMV and that of BaMMV consisted of 297 amino acids (891 nucleotides) and 251 amino acids (753 nucleotides), respectively. The snalysis of coat protein genes from these two viruses showed that 45.6% of nucleotides sequence ad 34.9% of amino acid in BaYMV were homologous to those in BaMMV.

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