• Journal of Bio-Environment Control
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• v.11 no.3
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• pp.139-143
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• 2002

Cotton Glutathione S-Transferase (GST: EC 2.5.1.18) was cloned and overexpressed in tobacco (Nicotiana tabacum) plants. Northern blot analysis confirmed the successful transformation of cotton gst gene in tobacco plant. Type I and Type ll transcript patterns were identified in transgenic tobacco plants and only Type I transcripts were discussed in this paper, The activity of GST in the type II transgenic plants was about 1.5-fold higher than those of the wild type and non-expresser by using 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione as the substrate. The expression of cotton GST in tobacco plants proved that Gh-5 could be translated into functional protein. Type II transgenic plants produced functional GST in the cells. The effects of cotton GST in the seedlings was evaluated by growing the control and transgenic seedlings at $15^{\circ}C$ in the growth chamber in the light. Overexpressors were grown well compared to the control plants (non-expressors). lo test far tolerance to salinity, seeds of Gh-5 overexpressors and the wild type Xanthi seedlings were grown at 0, 50, 100, 150, and 200 mM NaCl solution. Gh-5 transgenic seedlings showed higher growth rate over control seedlings on 50 and 100 mM NaCl solution. There was no difference in growth rate at 150 and 200mM NaCl concentration.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.135-141
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• 2003

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM -CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 ${\mu}$g/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 ${\mu}$g/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation from E. coli.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.138-143
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• 1997

We have designed and constructed a gene encoding novel high essential amino acid encoding protein(HEAAE). The resultant DNA fragment was tested for in vitro and in vivo expression and then cloned into plant expression vector pBI121, under the control of the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens, strain LBA4404, was subsequently transformed with this new construct and Nicotiana tabacum var. Xanthi transgenic plants were obtained. DNA analysis by Southern procedure confirmed the presence of the multi-copy number of genes in the transformed plants. Analysis of RNA and protein synthesized in these transgenic plants demonstrated the stable expression of this gene.

• Molecules and Cells
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• v.28 no.1
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• pp.57-65
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• 2009

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57-83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.

• Journal of Plant Biology
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• v.38 no.2
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• pp.203-209
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• 1995

Tobacco (Nicotiana tahacum L. cv. Wisconsin 38) leaf discs were cocultivated with Agrohacterium carrying a leghemoglobin (Lb) cDNA from Canavalia lineata. Seven plants were regenerated from the transformed leaf discs on MS media supplemented with 0.5 mg/L BAP, 0.1 mg/L ${\alpha}-NAA$, 200 mg/L kanamycin and 500 mg/L carbenicillin. Southern hybridization and PCR of genomic DNA from transgenic plants showed that the Lb cDNA was stably integrated into the genome of the tobacco. Total RNA from the transgenic tobacco showed northern hybridization signal at 1,000 nt and PCR of the first strand cDNA synthesized from the total RNA amplified 0.5 kb Lb cDNA. Furthermore, western hybridization using a polyclonal antibody against soybean Lb showed a 15.8 kD LB-like band on SDS-PAGE of proteins from the transformed tobacco. These results demonstrated that the Lb cDNA of C. lineata was not only incorporated into the genome of tobacco, but also transcribed into mRNA and translated into Lb protein in the transformed tabacco.

• Journal of Life Science
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• v.14 no.4
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• pp.657-663
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• 2004

Tobacco transformants harboring cinnamic acid 4-hydroxylase gene (C4H) fused with susquiterpene cyclase promoter was developed in order to regulate biosynthesis of phenolic compounds by the expression of the introduced gene. Twenty transformants for each specific promoter were used to analyze the incorporation of the chimeric genes by PCR and Southern blot analysis. PCR products of NPTII(neomycin phosphotransferase) gene (553bp) were detected in the transgenic tobacco plants. The incorporation of the chimeric gene was confirmed in the Southern blot analysis. C4H activity in the transgenic plants was elevated by UV-irradiation and its level was higher compared to that of control plants.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.89-93
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• 1998

Tomato phenylalanine ammonia-lyase 5 (tPAL5) was identified that alternate initiation sites were utilized differentially in response to environmental stimuli (Lee et al, 1992b). In this study, we tried to look into tissue -or cell- specific expression pattern of tPAL5 gene by fusing with ${\beta}-glucuronidase$ (GUS) gene in transgenic tobacco plants. In transgenic plants, root and stem extracts contained 8~12 fold higher levels of GUS activity than petiole or leaf tissue while the highest levels of induction was observed from leaf tissue by mechanical wounding (5~11 fold). In trans-sections of stems and petioles, GUS activity was restricted to phloem cells(outer region) of developing vascular bundle and mainly at apical tip region in the root tissues. The levels of GUS activity was drastically reduced (10~12 fold reduction) when the 5'-upstream region of tPAL5 gene (-1151bp from ATG codon) was deleted up to -665. The levels of GUS expression, however, raised up by 6~8 fold when deleted up to -455. Therefore, we conclude that there are positive cis-elements at the region -1151 to -1008 and at -455 to -195 while the negative cis-element is at -1008 to -455.

• Horticultural Science & Technology
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• v.31 no.6
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• pp.748-755
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• 2013

This study was conducted to find a salt tolerance gene in Brassica rapa. In order to meet this objective, we analyzed data from a KBGP-24K oligo chip [BrEMD (Brassica rapa EST and microarray database)] of the B. rapa ssp. pekinensis 'Chiifu' under salt stress (250 mM NaCl). From the B. rapa KBGP-24K microarray chip analysis, 202 salt-responsive unigenes were primarily selected under salt stress. Of these, a gene with unknown function but known full-length sequence was chosen to closely investigate the gene function. The selected gene was named BrSSR (B. rapa salt stress resistance). BrSSR contains a 285 bp open reading frame encoding a putative 94-amino acid protein, and a DUF581 domain. The pSL94 vector was designed to over-express BrSSR, and was used to transform tobacco plants for salt tolerance analysis. T1 transgenic tobacco plants that over-expressed BrSSR were selected by PCR and DNA blot analyses. Quantitative real-time RT PCR revealed that the expression of BrSSR in transgenic tobacco plants increased by approximately 3.8-fold. Similar results were obtained by RNA blot analysis. Phenotypic characteristics analysis showed that transgenic tobacco plants with over-expressed BrSSR were more salt-tolerant than the wild type control under 250 mM NaCl for 5 days. Based on these results, we hypothesized that the over-expression of BrSSR may be closely related to the enhancement of salt tolerance.

• Journal of Life Science
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• v.18 no.3
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• pp.304-310
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• 2008

Heat shock protein 70 (HSP70) is known as molecular chaperone, the fundamental protein participating in various processes, from nascent protein synthesis to protection of proteins during abiotic stresses and developmental programs. However, their biological functions in plants are not yet well known. Here, NtHSP70-1 (AY372069), HSP70 of Nicotiana tabacum induced by heat stress was investigated. To analyze the protective role of NtHSP70-1, transgenic tobacco plants, which constitutively overexpressed NtHSP70-1 as well as contained either the vector alone or having NtHSP70-1 in the antisense orientation, were constructed. The altered NtHSP70-1 levels in plants were confirmed by western blotting and transgenic sense lines exhibited tolerance to heat stress. Seedlings with the constitutively expressed NtHSP70-1 grew as green or healthy plants after heat stress. In contrast, transgenic vector or antisense lines exhibited yellowing of leaves or some delay in growth, which finally led to death. Evaluation of chlorophyll contents of heat-shocked transgenic tobacco seedlings indicated that NtHSP70-1 contributes to thermotolerance by preventing chlorophyll synthesis in plants.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.9-14
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• 2006

As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.