• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.390-394
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• 2004

The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.77-83
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• 1999

Molecular biological techniques that recently developed, have made it possible to realize some of new attempts in the research field of rumen microbiology. Those are 1) cloning of genes from rumen microorganisms mainly in E. coli, 2) transformation of rumen bacteria and 3) ecological analysis with nonculturing methods. Most of the cloned genes are for polysaccharidase enzymes such as endoglucanase, xylanase, amylase, chitinase and others, and the cloning rendered gene structural analyses by sequencing and also characterization of the translated products through easier purification. Electrotransformation of Butyrivibrio fibrisolvens and Prevotella ruminicola have been made toward the direction for obtaining more fibrolytic, acid-tolerant, depoisoning or essential amino acids-producing rumen bacterium. These primarily required stable and efficient gene transfer systems. Some vectors, constructed from native plasmids of rumen bacteria, are now available for successful gene introduction and expression in those rumen bacterial species. Probing and PCR-based methodologies have also been developed for detecting specific bacterial species and even strains. These are much due to accumulation of rRNA gene sequences of rumen microbes in databases. Although optimized analytical conditions are essential to reliable and reproducible estimation of the targeted microbes, the methods permit long term storage of frozen samples, providing us ease in analytical work as compared with a traditional method based on culturing. Moreover, the methods seem to be promissing for obtaining taxonomic and evolutionary information on all the rumen microbes, whether they are culturable or not.

• Plant Biotechnology Reports
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• 제2권3호
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• pp.219-226
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• 2008

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient ${\beta}-glucuronidase$ (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and $100{\mu}F$ in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1-2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

• International Journal of Industrial Entomology and Biomaterials
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• 제25권2호
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• pp.187-193
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• 2012

Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5- and 3- end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.

• 한국초지조사료학회지
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• 제20권2호
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• pp.97-102
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• 2000

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants via Agrobacterium-mediated gene transfer system. Calli induced from scutella were co-cultivated with a A. tumefaciens strain EHAlOl canying a plasmid, pIGhsp21. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of Oshsp2l gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot and immunoblot analyses revealed that the Oshsp21 gene was constitutively expressed and accumulated as mature protein in transgenic plants. Effects of constitutive expression of the OshspZl on thermotolerance were first probed with the chlorophyll fluorescence. Results indicate that inactivation of electron transport reactions in photosystem I1 (PSII), were mitigated by constitutive expression of the Oshsp21. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery during heat stress. (Key words : Thermotolerance, Rice, Transgenic, cDNA)

• 한국미생물·생명공학회지
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• 제18권6호
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• pp.647-652
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• 1990

Bacillus thuringiensis subsp. kurstaki HD1의 내 독소생산과 내독소단백질 유전자의 클로닝에 관한 연구를 하였다. 상기 균주는 아포생성기간 중에 이중피라미드형의 내독소를 생산하였고, 크리는 약 $2.9\times 1.0 \mu m$이었다. 상기 균주는 약 10개의 플라수미드 DNA를 가지고 있었으며, 플라스미드의 분자량의 범위는 2.1에서 80kilobases였다. 플라스미드 73kb, BamHI 절단 29Kb DNA 단편과 PstI 절단 7.9Kb DNA는 Probe DNA와 혼성화되었다. PstI 7.9Kb DNA를 추출하여 운반체인 pBR322 운반체의 PstI 절단부위에 삽입하여 클로닝한 후에 E.coli HB101 균주에 형질전환하였으며, 이 클로운을 pKL-20-1로 명명했고 이 형질전환체는 Bombyxmori 유충을 치사시키는 독소물질을 생산하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권9호
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• pp.1331-1341
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• 2001

Dairy cows are prone to fatty liver during the time of periparturient. Despite of the extensive studies, etiology and solutions for fatty liver are still not well known.The liver synthesizes triglycerides (TG) using precursors from bloodstream and secretes TG in form of very low density lipoprotein (VLDL) into bloodstream for the utilization by peripheral tissues. When the amount of TG synthesis exceeds the amount of secretion in VLDL-TG, TG accumulation within the liver occurs. Hepatic VLDL assembly and secretion involve multi-biochemical events.The availabilities of apolipoprotein B (apoB), E (apoE), microsomal triglyceride transfer protein (MTP) and soluble low density lipoprotein (LDL) receptor are now believed to be some of the main regulators for hepatic VLDL assembly and secretion. Studies in transgenic animals show that overexpression of these proteins stimulates VLDL production and secretion, which provides a possibility for alleviating bovine fatty liver by gene therapy. However, many problems remain to be solved to attain this goal. This review focuses on the molecular mechanisms of hepatic VLDL assembly and secretion, and the possibilities and problems of applying the knowledges to solve bovine fatty liver by gene therapy.

• Asian-Australasian Journal of Animal Sciences
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• 제4권1호
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• pp.73-78
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• 1991

Sixty-one 5-11 month-old California, Chinchilla and New Zealand White rabbit were employed in this investigation. Thirty-three does were superovulated by injecting FSH/HCG subcutaneously or intravenously and then sacrificed at different hours after mating. The ova were collected from the fallopian tubes with Ham's F-10 medium supplemented with 0.4% bovine serum albumin (BSA) and 1% pregnant rabbit serum (PRS). Embryos were examined under an inverted DIC microscopy for observing the stage of development. We have found that the fertilized ova formed pronuclei at 19 - 20 hr postcoitus. Approximately at 26, 64 - 78 and 84 - 88 hr after mating, the fertilized ova cleaved further to 2-cell, morulae and blastocyst stage respectively. Another 28 does were allocated to the gene transfer study. Fourteen of the 28 does were sacrificed at 19 - 20 hr to donate the pronuclear stage ova for gene injection. The other 14 does were induced to pseudopreganacy by injection of 100 IU HCG intravenous as recipients. Four hundreds and seventeen ova were injected totally and 212 gene injected zygotes were transferred into the recipient oviducts. Five recipients became pregnant and 10 fetuses were obtained. Eight of the 10 fetuses were analysed for gene incorporation, but none of them were transgenic.

• Plant Biotechnology Reports
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• 제2권1호
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• pp.47-58
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• 2008

To select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, two-step scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.