• Molecules and Cells
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• v.19 no.1
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• pp.81-87
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• 2005

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.

• 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.

• The Plant Pathology Journal
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• v.31 no.4
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• pp.433-437
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• 2015

The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.169-173
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• 2000

A 1,183bp cDNA, AmA1, encoding the seed storage protein of Amaranthus hypochondriacus was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) and characterized. AmA1 gene was subcloned into plant binary vector under Cauliflower Mosaic Virus (CaMV) 35S promoter and nopaline synthase terminator (3'NOS). The recombinant binary vector was used to transform Nicotiana tabacum using Agrobacterium tumefacien -mediated transformation procedure. Shoots were induced on MS medium with 0.1 mg/L NAA, 1.0 mg/L BA, 100 mg/L kanamycin and 250 mg/L cefotaxime. Transgenic plants were selected on rooting medium based on MS medium containing 200 mg/L kanamycin and 250 mg/L cefotaxime without phytoregulators. The presence of AmA1 gene in the transgenic plants was confirmed by PCR followed by DNA hybridization. The expression of AmA1 gene in the transgenic plant was observed by RT-PCR method.

• Journal of Plant Biotechnology
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• v.43 no.4
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• pp.432-437
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• 2016

Interleukin-11 (IL-11) is a cytokine that plays a key regulatory role in the immune system. Recombinant human IL-11 (rhIL-11) exerts a preventative effect against apoptotic cell death and inhibits preadipocyte differentiation. IL-11 also is used to stimulate the bone marrow to produce platelets in order to prevent low platelets that may be caused by chemotherapy. Unfortunately, the high production cost of IL-11 associated. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-11. Production of rhIL-11 proteins in whole-plant expression system will be more economical when compared to the current E. coli based expression system. The human rhIL-11 gene was codon optimized to maximize plant host system expression. IL-11 expression vector under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into tobacco by Agrobacterium-mediated transformation. The 5'-leader sequence (called ${\Omega}$) of tobacco mosaic virus (TMV) as a translational enhancer was added to construct. Transgenic tobacco plants expressing various levels of rhIL-11 protein were generated. Western blotting of the stably transformed lines demonstrated accumulation of the appropriately sized rhIL-11 protein in leaves. This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11.

• BMB Reports
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• v.40 no.3
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• pp.396-403
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• 2007

Proline accumulation has been shown to correlate with tolerance to drought and salt stresses in plants. We attempt to introduce the wild-type, mutant, and fusion proBA genes derived from Bacillus subtilis into Arabidopsis thaliana under the control of a strong promoter cauliflower mosaic virus 35S (CaMV35S). The transgenic plants produced higher level of free proline than control and the overproduction of proline resulted in the increased tolerance to osmotic stress in transgenic plants. Besides, the mutation in proBA genes, which were proved to lead $\alpha$-glutamyl kinase ($\alpha$-GK) reduces sensitivity to the end-product inhibition and the fusion of proB and proA also result in increasing proline production and confer osmotolerance in transgenic lines.

• Journal of Plant Biotechnology
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• v.50
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• pp.56-62
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• 2023

Transgenic lilies have been obtained using Agrobacterium tumefaciens (AGL1) with the plant scale explants, followed by DL-phosphinothricin (PPT) selection. In this study, scales of lily plants cv. "red flame" were transformed with the pCAMBIA3301 vector containing the gus gene as a reporter and the blpR gene as a selectable marker, as well as a gene of interest showing herbicide tolerance, both driven by the CaMV 35S promoter. Using a 20-minute infection time and a 5-day cultivation period, factors that optimized and demonstrated a high transformation efficiency were achieved. With these conditions, approximately 22-27% efficiency was observed for Agrobacterium-mediated transformation in lilies. After transformation with Agrobacterium, scales of lilies were transferred to MS medium without selective agents for 2 weeks. They were then placed on selection MS medium containing 5 mg/L PPT for a month of further selection and then cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets after transferring into hormone-free MS medium. Also, most survived putatively transformed plantlets indicated the presence of the blpR gene by PCR analysis and showed a blue color indicating expression of the gus gene. In conclusion, when 100 scales of lily cv. "red flame" are transformed with Agrobacterium, approximately 22-27 transgenic plantlets can be produced following an optimized protocol. Therefore, this protocol can contribute to the lily breeding program in the future.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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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 Plant Biotechnology
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• v.37 no.3
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• pp.327-336
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• 2010

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage (Brassica rapa) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably integrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the $T_2$ and $T_3$ transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

• Journal of Plant Biotechnology
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• v.46 no.4
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• pp.282-290
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• 2019

Cuphea viscosissima plants accumulate medium-chain fatty acids (MCFAs), i.e., those containing 8 ~ 14 carbons, in their seeds, in addition to the longer carbon chain fatty acids (≥16 carbons) found in a variety of plant species. Previous studies have reported the existence of three C. viscosissima MCFA-producing acyl-acyl carrier protein (ACP) thioesterases with different substrate specificities. In this study, CvFatB4, a novel cDNA clone encoding an acyl-ACP thioesterase (EC 3.1.2.14), was isolated from developing C. viscosissima seeds. Sequence alignment of the deduced amino acid sequence revealed that four catalytic residues for thioesterase activity are conserved and a putative N-terminal chloroplast transit peptide is present. Overexpression of CvFatB4 cDNA, which was under the control of the cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana led to an increase in 16:0 fatty acid (palmitate) levels in the seed oil at the expense of 18:1 and other non-MCFAs.