• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.103-108
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• 2003

LinA gene involving in the ${\gamma}$-benzenehexachloride degradation have been cloned from Sphingmonas paucimobilis UT26. This linA gene which catalyzes the first dechlorination step of ${\gamma}$-benzenehexachloride is known to play a key role in the ${\gamma}$-benzenehexachloride degradation pathway in UT26. In this study, the linA gene was designed to clean-up the ${\gamma}$-benzenehexachloride and its derivatives contaminated in soil, water and air using transgenic tobacco plants. The linA transgene was introduced into the chromosome of tobacco using leaf-disk transformation approach as revealed by Southern blot analysis. In addition, mRNA and protein produced by linA gene was expressed at a high level in the leaf tissue as demonstrated by both northern blot analysis and Western bolt analysis with polyclonal antibody against S. paucimobilis UT26. in vitro analysis using GC-MS showed that transgenic tobacco plant produced the linA protein which effectively degraded ${\gamma}$-benzenehexachloride into ${\gamma}$- pentachlorocyclohexene and 1,2,4-trichlobenzene compounds which are less toxic.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.305-311
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• 1997

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

• Korean journal of applied entomology
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• v.54 no.1
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• pp.7-15
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• 2015

CpBV (Cotesia plutellae bracovirus) is a polydnavirus and encodes a cystatin (CpBV-CST1) gene. Its overexpression suppresses insect immunity and alters insect developmental processes. This study aimed to construct a genetically modified (GM) tobacco to further explore the physiological function of the viral cystatin and to apply to control insect pests. To this end, the transgenic tobacco lines were screened in expression of the target gene and assessed in insecticidal activity. A recombinant vector (pBI121-CST) was prepared and used to transform a bacterium, Agrobacterium tumefasciens. The transformed bacteria were used to generate transgenic tobacco lines, which were induced to grow callus and resulted in about 92% of shoot regeneration. The regenerated plants were screened by PCR analysis to confirm the insertion of the target gene in the plant genome. In addition, the expression of the target gene was assessed in the regenerated plants by quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis showed that the transgenic line plant expressed the target gene about 17 times more than the control tobacco, indicating a stable insertion and expression of the target gene in the transgenic tobacco line. The insecticidal activity was then analyzed using the screened transgenic tobacco lines against the teneral 1st instar larvae of the oriental tobacco budworm, Helicoverpa assulta. Though there was a variation in the insecticidal efficacy among transgenic lines, T9 and T12 lines exhibited more than 95% mortality at 7 days after feeding treatment. These results suggest that CpBV-CST1 is a useful genetic resource to be used to generate GM crop against insect pests.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04a
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• pp.49-58
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• 2002

Oxidative stress derived from reactive oxygen species (ROS) is one of the major damaging factors in plants exposed to environmental stress. In order to develop the platform technology to solve the global food and environmental problems in the 21s1 century, we focus on the understanding of the antioxidative mechanism in plant cells, the development of oxidative stress-inducible antioxidant genes, and the development of transgenic plants with enhanced tolerance to stress. In this report, we describe our recent results on industrial transgenic plants by the gene manipulation of antioxidant enzymes. Transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts were developed and were evaluated their protection effects against stresses, suggesting that simultaneous overexpression of both SOD and APX in chloroplasts has synergistic effects to overcome the oxidative stress under unfavorable environments. Transgenic tobacco plants expressing a human dehydroascorbate reductase gene in chloroplasts were showed the protection against the oxidative stress in plants. Transgenic cucumber plants expressing high level of SOD in fruits were successfully generated to use the functional cosmetic purpose as a plant bioreactor. In addition, we developed a strong oxidative stress-inducible peroxidase promoter, SWPA2 from sweetpotato (Ipomoea batatas). We anticipate that SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.149-155
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• 1995

The coat protein (CP) gene was cloned from RNA genome of the Cucumber Mosaic Virus strain ABI (CMV-ABI) isolated in Korea. The comparisons of the nucleotide sequence of the cloned CP gene and its deduced amino acid sequences with other CP genes revealed that the CMV-ABI belongs to subgroup I (type I), CMV-ABI developed the typical mosaic symptom in infected plants. Tobacco plants (Samsun and NC82) were transformed by leaf-disc transformation via Agrobacterium, temefaciens LB4404 harboring pVCP, witch CMV-ABI CP gene was inserted into the pBI121, and a number of mature transgenic tobacco plants were developed. Southern and PCR analysis of genomic DNA from the transgenic plants showed that the CP gene was integrated into the genomes of the most of the transgenic plant. Result of the segregation patterns of resistance in T1 seedlings of the plants to kanamycin showed that the transgenic plants containing l,2 and 3 copies of CP gene were50%, 39% and 11% of the total transgenic plants, respectively.

• BMB Reports
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• v.41 no.5
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• pp.376-381
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• 2008

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.

• 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 Environmental Science International
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• v.25 no.1
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• pp.173-179
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• 2016

Leaf water and osmotic potential, chlorophyll content, photosynthetic rate, and electrolyte leakage were measured to evaluate tolerance to water stress in wild-type (WT) and transgenic tobacco plants (TR) expressing copper/zink superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) in chloroplasts. Leaf water potential of both WT and TR plants decreased similarly under water stress condition. However, leaf osmotic potential of TR plants more negatively decreased in the process of dehydration, compared with WT plants, suggesting osmotic adjustment. Stomatal conductance (Gs) in WT plants markedly decreased from the Day 4 after withholding water, while that in TR plants retained relatively high values. Relatively low chlorophyll content and photosynthetic rate under water stress were shown in WT plants since $4^{th}$ day after treatment. In particular, damage indicated by electrolyte leakage during water stress was higher in WT plants than in TR plants. On the other hand, SOD and APX activity was remarkably higher in TR plants. These results indicate that transgenic tobacco plants expressing copper/zink superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) in chloroplasts improve tolerance to water stress.

• Journal of The Korean Society of Grassland and Forage Science
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• v.22 no.2
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• pp.101-106
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• 2002

This study was conducted to obtain the transformed tobacco (Nicotiana tubacum) plants with cytosolic ascorbate peroxidase gene(ApxSC7) using Agrobacterium tumefaciens LBA4404. A cDNA encoding the cytosolic ascorbate peroxidase of strawberry, ApxSC7, was introduced into tobacco plants via Agrobacterium-mediated gene transfer system. The expression vector, pIG-AP8, harboring ApxSC7 gene was used for production of transgenic tobacco plants. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of ApxSC7 gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot analyses revealed that the pIGap8 gene was constitutively expressed.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.195-200
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• 1995

The mammalian adenosine deaminase(ADA) gene was stably expressed in transgenic tobacco plane. The chimeric ADA gene 35S/35S/AMV/ADA/Tnos, has been constructed. This chimeric gene was introduced into the binary vector pRD400, which was thereafter mobilized into Agrobacterium tumefaiens strain MP90 harboring disarmed Ti-plasmid. The resulting strains were used to transform Nicofiana tabacum L. using the leaf disc. Incorporation of the chimeric gene into plant were confirmed by PCR and Northern blot analyses. Immunoblot analysis showed that ADA protein was successfully synthesized in the transgenic tobacco plants.