• Journal of Plant Biotechnology
• /
• v.38 no.1
• /
• pp.42-48
• /
• 2011

Chloroplast genetic engineering of higher plants offers several unique advantages compared with nuclear genome transformation, such as high levels of transgene expression, a lack of position effect due to site-specific transgene integration by homologous recombination, multigene engineering in a single transformation event and reducing risks of gene flow via pollen due to maternal inheritance. We established a reproducible chloroplast transformation system of potato using a tobacco specific plastid transformation vector, pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA). Stable transgene integration into chloroplast genomes and the homoplasmic state of the transgenome were confirmed by PCR and Southern blot analyses. Northern, immunoblot analysis, and GFP fluorescence imaging revealed high expression and accumulation of GFP in the plastids of potato leaves. This system would provide new opportunities for genetic improvement and mass production of value added foreign proteins in this crop.

• Horticultural Science & Technology
• /
• v.18 no.5
• /
• pp.594-598
• /
• 2000

This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.27 no.2
• /
• pp.129-134
• /
• 2001

Since NNK is one of the most abundant tobacco-specific alkaloids and a strong carcinogenic nitrosamine, it has been used for evaluating a potential of carcinogenicity in the animal models. The present study has attempted to examine the potential of carcinogenicity of NNK in human epithelial cells, from which the cell type the most of cancers including oral cancer and nasal cavity cancer are originated. The cellular model used for the study is a human keratinocyte cell system immortalized by Ad12-SV40 hybrid virus. The cellular system has successfully been used for the carcinogenicity studies because of its limitless life span, epithelial morphology and nontumorigenicity. When cells were treated with a variety of NNK concentrations, levels of saturation density and soft agar colony formation were increased in a dose-dependent fashion. Colonies of large cell aggregates were above 5 at the higher doses. The results indicate that exposure of human cells with NNK induced loss of contact inhibition and increases of anchorage independence and cellular adhesion, which are typical characteristics of the neoplatically transformed cells. When cells were exposed with 100uM NNK for 2hr, mRNA levels of IL-1 and PAI-2 were increased in a dose-dependent manner, but expression of TGF- 1 was not affected. While expression of growth regulatory factors were altered with a short-term exposure, there was no alteration of these factors in the NNK-transformed cells. However, mRNA levels of fibronectin were increased both in the short-term treatment and in the transformation. The results suggest that altered expression of extracellular matrix such as fibronectin following short-term exposure might be fixed in the genome and these altered properties be continuously transfered throughout the cell division. Western blot analysis showed a translocation of PKC- from cytosolic fraction to the particulate fraction, indicating a possible role of NNK in the signal transduction pathway. The present study provided an evidence that NNK in the smoking may be associated with epithelial origin cancer such as oral and nasal cavity cancers. In addition, this study suggested that altered expression of extracellular matrix and PKC may play an important role in the carcinogenic mechanism of NNK.

• Horticultural Science & Technology
• /
• v.17 no.4
• /
• pp.473-475
• /
• 1999

This study was carried out to restore the fertility by suppression of male sterility-induced gene using an antisense construct. Tobacco (cv. Petit Havana SR1) was transformed with the binary vector containing a GBAN215-6 promoter, an antisense diphtheria toxin (DTx-A) gene (pKDA215b) and a hygromycin resistant gene. Seventy-six confirmed transgenic plants regenerated from leaf disks were designated as the $R_0$ generation and selfed to produce the $R_1$ generation. From the inheritance study, five $R_1$ lines with multiple copies of the antisense construct were selected and selfed to identify homozygosity for the antisense construct. In order to restore fertility and finally to select restore lines, five $R_2$ lines with multiple copies of the antisense construct were crossed with male sterile plants. From these crosses, three different phenotypes have been observed: completely restored, partially restored, and not restored pollens, and otherwise tobacco plants were phenotypically same as normal plants. These plants were scored for the degree of restoration and selected for further study.

• Korean Journal of Plant Tissue Culture
• /
• v.24 no.1
• /
• pp.63-69
• /
• 1997

Erythropoietin (EPO) is a glycoprotein that mediates the growth and differentiation of erythroid progenitors. In order to produce recombinant human erythropoietin in tobacco plant, the EPO genomic DNA (5.4 kb) was cloned into plant expression vectors, pBI$\Delta$GUS121, pBD$\Delta$GUS121 and pPEV-1, and introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated transformation. After selection on MS media containing kanamycin (Km), 10 Km-resistant plants were obtained per each construct. The correct integration of EPO genomic DNA in the genome of transgenic plant was confirmed by polymerase chain reaction (PCR). Northern blot showed that transcripts of 1.8 kb length were produced in leaves of the plants, but there was no difference of mRNA amount according to promoter number and 5'-untranslated sequence (UTS). The proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbit anti-human EPO polyclonal antibody. The expressed protein appeared as smaller band of apparent mass of 30 kDa as compared to the EPO protein from human urine (37 kDa), suggesting that the modification (glycosylation) system in tobacco plant might be different from that of mammalian cells.

• Journal of Chest Surgery
• /
• v.36 no.9
• /
• pp.666-673
• /
• 2003

Cigarette smoking is the leading cause of the lung cancer. However, mechanism of action underlying the carcinogenesis in the lung still remains to be elucidated. The present study attempted to look into the carcinogenic potential of tobacco-specific nitrosamine, NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and the effects of protein kinase C (PKC) isoforms in an immortalized human epithelial cell model. Material and Method: Immortalized human epithelial cells were exposed with NNK and examined for its carcinogenic potential as measured by saturation density, soft-agar colony formation, and cell aggregation assay. The specific isoform of PKCs involved in the cellular transformation was analysed through western blot with monoclonal antibody and measured separately in cytosolic fraction and membrane fraction. Result: Human epithelial cells exposed with NNK showed prominent carcinogenic potential in saturation density, soft agar colony formation, and cell aggregation assay. PKC isoform analysis results are as follows: PKC- $\alpha$ showed significant translocation of protein levels from cytosolic fraction to membrane fraction, as analyzed by immunoblot. PKC- $\varepsilon$ showed a dose-dependent increase of translocation. PKC- λ was not affected by NNK treatment. Conclusion: The study demonstrated that there was a certain specificity in the patterns of isoform induction following chemical carcinogen exposure. Thus, it is suggested that identification of specific isoform be a clue to find target molecules in the carcinogenesis.

• Journal of Plant Biotechnology
• /
• v.38 no.1
• /
• pp.54-61
• /
• 2011

Antisense construct of cDNA for senescencerelated ACC oxidase (CAO) cDNA isolated from carnation flowers were introduced into tobacco by Agrobacteriummediated transformation. The decreasing expression of NtACO and the reduction of ethylene production were observed in these transgenic lines. In contrast, the SAMDC transcripts and spermidine content were increased. The findings that higher content of spermidine in the ethylene suppressed transgenic plants compared with wild-type should be directly resulted in the enhancement of SAMDC activity followed by the increased accumulation of SAMDC transcript. To investigate the pathogenic response in these transgenic plants, wild-type and transgenic plants were inoculated with Phytophthora parasitica pv. nicotianae. Transgenic plants suppressing ethylene production showed the increased resistance against fungal pathogen, comparing with wild-type plant. PR-protein genes expression in CAO-AS-2 and CAOAS-4 were also higher at the normal growth condition and pathogenic response than in wild-type plants. The results of higher spermidine content and SAMDC activity in transgenic plants, CAO-AS-2 and CAO-AS-4, support the possibility that an increase in spermidine content might induce the higher transcripts of PR-protein genes. This results agreed with the phenomena that spermidine promoted the expression of PR1a and a SAMDC inhibitor, MGBG, decreased the expression of PR1a in leaf discs. These results suggest that the resistance against fungal pathogen in transgenic tobacco impaired in ethylene production might be caused by increasing in polyamine, especially spermidine, biosynthesis.

• Korean Journal of Plant Tissue Culture
• /
• v.21 no.6
• /
• pp.353-356
• /
• 1994

Chimeric kappa chain and gamma chain cDNA clones (pCKS2 and pCHS2) of a monoclonal antibody specific for pre-S2 surface antigen of human hepatitis-B virus were ligated into Xbal site of plant expression vector pBKS-1. Plasmid DNA containing each of the chimeric gene were then mobilized from E, coli to Agrobacterium tumefaciens strain LBA4404. The chimeric antibody genes were then introduced into tobacco by Ti plasmid-mediated transformation. The putative Transformants were selected on medium containing kamaycin sulfate. Shoots that formed on shoot induction medium were analyzed by Western blot analysis for the expression of kappa-chain or gamma-chain genes. The Western blot analyses clearly showed that the introduced genes were stably expressed in transgenic plants.

• Journal of Plant Biotechnology
• /
• v.37 no.4
• /
• pp.567-573
• /
• 2010

To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

• Plant Biotechnology Reports
• /
• v.2 no.3
• /
• pp.199-206
• /
• 2008

We have developed an efficient system of assessing the ability of a gene silencing cassette to silence transcripts from recalcitrant or poorly studied plant species by using a model plant as a host for the gene of interest. Tobacco plants transgenic for Lachrymatory Factor Synthase (LFS) enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. In this work, we have demonstrated that the RNAi construct is a suitable candidate for the development of a non-lachrymatory onion. Our model plant RNAi system has wide-reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in the pharmacological, food and process industries.