• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.191-191
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• 2017

Hydroxy fatty acid (HFA) is an important industrial resource that known to be extracted from seeds of Castor or Lesquerella. However, mass production of HFA from those crops are difficult because of their behavior or life cycle. In this study, we applied HFA synthesis related gene FAH12, RcPDAT1, RcLPCAT, RcDGAT2, and RcPDCT on bioenergy crop Camelina sativa. Furthermore, we determined NaCl or cold stress tolerance changes of transgenic Camelina. RcFAH12, RcPDAT1, RcLPCAT, RcDGAT2, and RcPDCT genes were cloned into multigene expression vector which is engineered with seed specific promoter of FAE1 or Napin. Combination of HFA genes multi-expression vector constructs were divided into Set3 (RcFAH12, RcPDAT1-2, RcLPCAT), Set4 (RcFAH12, RcDGAT2, RCPDAT1-2, RcLPCAT), and Set5 (RcFAH12, RcDGAT2, RCPDAT1-2, RcLPCAT, RcPDCT). Transgenic HFA synthesis Camelina plants were generated using agrobacterium-mediated vacuum infiltration system. Results of fatty acid composition of T1 transgenic Camelina seeds analyzed by GC-MS showed 9.5, 9.0, and 13.6 % of HFA contents in Set3#6, Set4#8, and Set5#10, respectively. Therefore, seeds of T2 generation were harvest from Set5#10 which is shown highest HFA contents, and, 17.7, 8.1 and 10.5 % of HFA contents were determined in Set5#10-5, Set5#10-8, and Set#10-10, respectively. However, 7.7% of C18:2 and 22.3 % of C18:3 among unsaturated fatty acids were decreased in Set5#10-5 than WT. Meanwhile, we confirmed abiotic stress responses in T2 transgenic Camelina Set5#10-5 and Set5#10-10 under 0, 100, 150, and 200 mM NaCl or 25, 15, and $10^{\circ}C$ temperature for 5 weeks. Both Set5#10-5 and Set5#10-10 showed lower growth in height than WT in control and NaCl condition. Growth of leaf length and width were similar in WT and Set5#10-10 but lower in Set5#10-5 under NaCl stress. Number of opened flowers showed that both transgenic Camelina were lower than WT under normal condition. But, WT and Set5#10-10 showed similar opened flower number in 100 and 200 mM NaCl. In cold stress, 15 and $10^{\circ}C$ treatment for 5 weeks did not showed significant changes in between WT and both transgenic lines even they showed different growth rate in control condition. Taken together, growth and development are delayed by expression of exogenous HFA related genes in transgenic lines but relative abiotic stress sensitivity is similar with WT. In conclusion, reduced C18:2 or C18:3 fatty acid composition of seed by HFA synthesis is resulted from lack of resource supplement for development at seedling stage but it is not affect NaCl and cold stress tolerance.

• Journal of Life Science
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• v.27 no.3
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• pp.295-300
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• 2017

This study examined the association between genotypes of the isocitrate dehydrogenase 3, beta subunit (IDH3B) gene and economic traits in an $F_2$ population of Duroc and Jeju (South Korea) native black pigs (JBPs). The genotypes was determined the presence/absence of a 304-bp insertion/deletion fragment in the promoter region of the IDH3B gene for JBP, Duroc, and their $F_1$ and $F_2$ progeny. Three genotypes (AA, AB and BB) were found in the $F_1$ and $F_2$ populations, but there was no AA genotype found in JBP and no BB in Duroc. Association analysis results showed the significant differences with carcass weights (CW), backfat thicknesses (BFT) and eye muscle area (EMA) (p<0.05), but not with growth traits including body weights and average daily gains at different stages, reproductive traits including teat numbers, and crude fat contents (CFAT) measured in longissimus dorsi (p>0.05). The $F_2$ pigs possessing the IDH3B BB homozygote had heavier CW ($72.92{\pm}11.133kg$), thicker BFT ($25.75{\pm}6.06mm$), and larger EMA ($23.82{\pm}4.825cm^2$) than those from the other genotypes (p<0.05). These results were estimated that there are biological roles related with IDH3B genotypes resulting development of EMA, BFT, and CW but not with intramuscular fat deposition during late period of pig production. Our findings suggest that the 304-bp insertion allele of porcine IDH3B may be a genetic marker for marker assistant selection for improving meat productivity of the Jeju Black pig and Duroc-related molecular breeding systems.

• Journal of Plant Biotechnology
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• v.40 no.4
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• pp.210-216
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• 2013

Efficient regeneration methods and transformation system are a priority for successful application of genetic engineering to vegetative propagated plants such as grape (Vitis labrusca L.). This research is to establish shoot regeneration system from plant explants for 'Campbell Early', 'Tamnara', 'Heukgoosul', 'Heukbosek' using two types of plant growth regulators supplemented to medium. The highest adventitious shoot regeneration rate of 5% was achieved on a medium containing of Murashige and Skoog (MS) inorganic salts and Linsmaier and Skoog (LS) vitamins, 2 mg/L of TDZ and 0.1 mg/L of IBA. Leaf tissue of 'Campbell Early', was co-cultivated with Agrobacterium strains, LBA4404 containing the vector pBI121 carrying with CaMV 35S promoter, gus gene as reporter gene and resistance to kanamycin as selective agent, the other Agrobacterium strains, GV3101 containing the vector pB7 WG2D carrying with mPAP1-D gene. mPAP1-D is a regulatory genes of the anthocyanin biosynthetic pathway. 'Campbell Early' harboring mPAP1-D gene was readily able to be selected by red color due to anthocyanin accumulation in the transformed shoot. These results might be helpful for further studies to enhance the transformation efficiency in grape.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.112-118
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• 2002

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by matting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB(spo5)1. We futher analyzed six recombinant plasmids, pDB(spo5)2, pDB(spo5)3, pDB(spo5)4, pDB(spo5)5, pDB (spo5)6, pDB(spo5)7 and found each of these plasmids is able to rescue the spo5-2, spo5-3, spo5-4, spo5-5, spo5-6, spo5-7 mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB(spo5)1, and pDB(spu5)Rl contained the spo5 gene. Transcripts of spo5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 2.5kb were detected with 5kb Hind Ⅲ fragment containing a part of the spo5 gene as a probe. The small mRNA(2.5kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2kb) was produced constitutively. Appearance of a 2.5kb spo5-mRNA depends upon the function of the meil, mei2 and mei3 genes.

• Journal of Life Science
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• v.21 no.12
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• pp.1666-1677
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• 2011

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.

• Journal of Life Science
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• v.19 no.7
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• pp.949-955
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• 2009

Mucosal epithelia sense external stress signals and transmit them to the intracellular cascade responses. Ribotoxic stress-producing chemicals such as deoxynivalenol (DON) or other trichothecene mycotoxins have been linked with gastrointestinal inflammatory diseases by Fusarium-contamination. The purpose of this study was to test the hypothesis that DON evokes the epithelial sentinel signals of RNA-dependent protein kinase (PKR) and early growth response gene 1 (EGR-1), which together contribute to the pro-inflammatory cytokine interleukin 8 (IL-8) in human intestinal epithelial cells. PKR suppression by the dominant negative PKR expression attenuated DON-stimulated interleukin-8 production. Moreover, 1L-8 transcriptional activation by DON was also reduced by PKR inhibition in the human intestinal epithelial cells. Treatment with the PKR inhibitor also suppressed EGR-1 promoter activity, mRNA and protein induction, although mitogen-activated protein (MAP) kinases such as extracellular signal-regulated protein kinases (ERK) 1/2, p38, c-Jun N-terminal Kinase (INK) were little affected or even enhanced in presence of a PKR inhibitor. These patterns were also compared in the EGR-1-suppressed cells, which showed much more suppressed production of 1L-8. All things taken into consideration, DON-activated sentinel signals of EGR-1 via PKR mediated interleukin-8 production in human intestinal epithelial cells, which provide insight into the possible general mechanism associated with mucosal inflammation as an intestinal toxic insult by ribotoxic trichothecene mycotoxins.

• Journal of Chest Surgery
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• v.37 no.12
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• pp.967-977
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• 2004

Background: DNA methylation is one of the important gene expression mechanisms of the cell. When cytosine of CpG dinucleotide in promotor is hypomethylated, expression of some genes that is controlled by this promoter is altered. In this study, the author investigated the effect of DNA demethylating agent, 5-aza-2'-deoxycytidine (ADC), on the expressions of cancer antigen genes, MHC and B7 in 4 lung cancer cell lines, NCIH1703, NCIH522, MRC-5, and A549. Material and Method: After treatment of cell lines, NCIH1703, NCIH522, MRC-5 and A549 with ADC (1 uM) for 48 hours, RT-PCR was performed by using the primers of MAGE, GAGE, NY-ESO-1, PSMA, CEA, and SCC antigen gene. In order to find the optimal ADC treatment condition for induction of cancer antigen, we studied the effect of ADC treatment time and dose on the cancer antigen gene expression. To know the effect of ADC on the expression of MHC or B7 and cell growth, cells were treated with 1 uM of ADC for 72 hours for FACS analysis or cells were treated with 0.2, 1 or 5 uM of ADC for 96 hours for cell counting. Result: After treatment of ADC (1 uM) for 48 hours, the expressions of MAGE, GAGE, NY-ESO-1, and PSMA genes increased in some cell lines. Among 6 MAGE isotypes tested, and gene expression of MAGE-1, -2, -3, -4 and -6 could be induced by ADC treatment. However, CEA gene expression did not change and SCC gene expression was decreased by ADC treatment. Gene expression was generally induced 24 - 28 hours after ADC treatment and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC ADC teatment, and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC teatment in ADC-Free medium. Most gene expression could be induced at 0.2 uM of ADC, but gene expression increased dependently on ADC treatment dose. The expression of MHC and B7 was not increased by ADC treatment in all four cell lines, and the growth rate of 4 cell lines decreased significantly with the increase of ADC concentrations. Conclusion: Treatment of lung cancer cell lines with ADC increases the gene expression MAGE, GAGE and NY-ESO-1 that are capable of induction of cytotoxic T lymphocyte response. We suggest that treatment with 1 uM of ADC for 48 hours and then culturing in ADC-free medium is optimal condition for induction of cancer antigen. However, ADC has no effect on MHC and B7 induction, additional modification for increase of expression of MHC, B7 and cytokine will be needed for production of efficient cancer cell vaccine.

• Journal of Life Science
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• v.22 no.4
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• pp.447-455
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• 2012

Akt plays an important role in a variety of cellular physiologies such as growth, proliferation, and differentiation. In skeletal muscle, Akt has been implicated in regulating regeneration, hypertrophy, and atrophy. In this study, the role of Akt has been examined during skeletal muscle differentiation. Culturing C2C12 myoblasts under low serum (1% horse serum) and high density converted cell morphology from a round shape to an elongated and multi-nucleated shape. Morphological changes were initiated from day 2 of differentiation. In addition, the expression of both myogenin G and myogenin D was elevated from day 2 of differentiation. Skeletal muscle differentiation was abolished by silencing Akt1 or Akt2, but was significantly enhanced by the over-expression of either Akt1 or Akt2. The activation of Akt was observed from day 2 of differentiation and disappeared after day 7. The expression of kruppel-like factor 4 was observed from day 6 of differentiation. Moreover, this expression was blocked in cells silencing either Akt1 or Akt2. In addition, the promoter activity of kruppel-like factor 4 was significantly reduced in cells silencing Akt1 or Akt2. These results suggest that Akt regulates skeletal muscle differentiation through the regulation of kruppel-like factor 4 expression.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.153-159
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• 2009

Recombinant Escherichia coli BLR(DE3) harboring the hemA gene from Rhodobacter capsulatus under the control of a constitutive promoter, which we constructed previously, was used for the extracellular production of 5-aminolevulinic acid (ALA). The effects of several factors on ALA production were investigated in flask culture. ALA production by the recombinant E. coli was more efficient at $30^{\circ}C$ than $37^{\circ}C$. The glycine concentration had an important effect on cell growth. Glycine and succinic acid concentration of 5-10 and 10-20 g/L, respectively, resulted in high ALA production. In addition, the partial replacement of succinic acid by sodium glutamate increased the ALA production. The ALA production was inhibited by the presence of glucose in the medium. Using the optimal conditions, an ALA concentration of 8.2 g/L was achieved in jar fermentation without an added inducer or ALA dehydratase inhibitor; this is the highest reported concentration.

• The Korean Journal of Mycology
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• v.47 no.4
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• pp.359-371
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• 2019

To optimize the expression and secretion of ferritin protein associated with ion storage in the mushroom, Pleurotus eryngii, a recombinant secretion vector, harboring the ferritin gene, was constructed using a pPEVPR1b vector under the control of the CaMV 35S promoter and signal sequence of pathogen related protein (PR1b). The ferritin gene was isolated from the T-Fer vector following digestion with EcoRI and HindIII. The gene was then introduced into the pPEVPR1b secretion vector, and it was then named pPEVPR1b-Fer. The recombinant vector was transferred into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformants were selected on MCM medium supplemented with kanamycin and its expression was confirmed by SDS-PAGE and western blotting. Expression of ferritin protein was optimized by modifying the culture conditions such as incubation time and temperature in batch and 20 L airlift type fermenter. The optimal conditions for ferritin production were achieved at 25℃ and after incubating for 8 days on MCM medium. The amount of ferritin protein was 2.4 mg/g mycelia, as measured by a quantitative protein assay. However, the signal sequence of PR1b (32 amino acids) seems to be correctly processed by peptidase and ferritin protein may be targeted in the apoplast region of mycelia, and it might not be secreted in the culture medium. The iron binding activity was confirmed by Perls' staining in a 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in P. eryngii mycelia. Mycelium powder containing ferritin was tested as a feed additive in broilers. The addition of ferritin powder stimulated the growth of young broilers and improved their feed efficiency and production index.