• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.205-211
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• 2021

In order to test the functionality of Panax ginseng thioredoxin 1 (PgTRX1) isolated from fermented wild ginseng roots, a transient effect on physiological activity were performed over a short time frame using the Agrobacterium infiltration technique. The PgTRX1 gene isolated from fermented wild ginseng was confirmed to have a size of 579 bp, and the expression of PgTRX1 was the highest in the sample after 6 h of fermentation. As a result of constructing this gene and confirming the infiltration reaction mediated by Agrobacterium in tobacco leaves, it was found that the expression of the NbHSR203j gene was also induced as PgTRX1 expression increased. As a result of measuring the biological activity of the infiltration samples, the total phenol content increased by 35.45±1.84 to 49.01±1.84 ㎍ GAE/mL compared to the control, and the total flavonoid amount of 9.52±0.41 to 9.82±0.25 ㎍ QE/mL was slightly high. From these results, Agrobacterium-mediated PgTRX1 appears to be related to the hypersensitive response induction mechanism of plants and the production of secondary metabolites such as phenolic substances.

• The Plant Pathology Journal
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• 제24권3호
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• pp.296-304
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• 2008

The transient and rapid expression system of a foreign protein in planta is a very useful technique in biotechnology application. We have investigated optimum condition of Agrobacterium-infiltration technique in which expression level of foreign proteins were maximized without detrimental effects on plants using GFP and Cucumber mosaic virus 2b protein, which is known as an enhancer of gene expression and a suppressor of post-transcriptional gene silencing(PTGS). The optimum expression level of both RNA and protein of GFP with minimum leaf impairment was obtained at $OD_{600}$=0.2 of Agrobactrium inocula. The steady-state levels of GFP RNA and protein generally peaked at 3 and 7 days post-infiltration(dpi), respectively. In the presence of 2b, both the magnitude and duration of GFP expression was highly increased and we could detect GFP level until 17 dpi. On the other hands, the 2b-mediated higher accumulation of foreign proteins resulted in the repression of normal leaf growth, possibly due to the limitation of supply of energy or materials required for growth maintenance. Using this Agrobacterium-infiltration system with 2b and GFP, we tested a hypothesis for the threshold model of PTGS initiation. Four GFP transgenic lines of N. benthamiana, which shows different expression level of GFP were tested to determine the threshold level for PTGS initiation. Agrobacterium-infiltration of GFP into those GFP-transgenic plants resulted in the co-silencing of the transgenic GFP. It was found that very low concentration of Agrobacterium with GFP and GFP+2b($OD_{600}$=0.002-0.02) which could not phenotypically induce an additive GFP expression, was enough to trigger PTGS pathway in all GFP transgenic plants. This strongly indicates that each GFP-transgenic plant should be expressing the transgenic GFP at its own pre-determined level and there was no buffer zone of additive GFP-expression to the threshold. In other words, the PTGS seems to be immediately activated as a self-defensive mechanism if an internal balance of gene expression is broken.

• Journal of Plant Biotechnology
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• 제48권3호
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• pp.156-164
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• 2021

Spirodela polyrhiza, from the Lemnaceae family, are small aquatic plants that offer an alternative plant-based system for the expression of recombinant proteins. However, no turion transformation protocol has been established in this species. In this study, we exploited a pB7YWG2 vector harboring the eYFP gene that encodes enhanced yellow fluorescent protein (eYFP), which has been extensively used as a reporter and marker to visualize recombinant protein localization in plants. We adopted Agrobacterium tumefaciens-mediated turion transformation via vacuum infiltration to deliver the eYFP gene to turions, special vegetative forms produced by duckweeds to endure harsh conditions. Transgenic turions regenerated several duckweed fronds that exhibited yellow fluorescent emissions under a fluorescence microscope. Western blotting verified the expression of the eYFP protein. To the best of our knowledge, this is the first report of an efficient protocol for generating transgenic S. polyrhiza expressing eYFP via Agrobacterium tumefaciens-mediated turion transformation. The ability of turions to withstand harsh conditions increases the portability and versatility of transgenic duckweeds, favoring their use in the further development of therapeutic compounds in plants.