• Journal of Ginseng Research
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• v.33 no.3
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• pp.212-218
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• 2009

Triterpenoid saponins were synthesized in Panax ginseng C.A. Meyer via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoids are backbone and undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450 (CYP)-dependent monooxygenases, glycosyltransferase and other enzymes. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. A cDNA clone encoding a putative CYP gene was isolated from flower bud of ginseng and transformed into the plant(Nicotiana tabacum cv. Xanthi) and confirmed by PCR analysis. The CYP gene (PgCYP) contained an open reading frame(ORF) encoding mature protein of 500 amino acids. The expression of PgCYP were investigated in transgenic tobacco by reverse transcriptase-polymerase chain reaction (RT-PCR).

• Korean Journal of Medicinal Crop Science
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• v.13 no.1
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• pp.57-62
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• 2005

Salt-tolerant transgenic Panax ginseng plants were produced by introducing the SAL1 geue (3'(2'), 5'-bis-phosphate nucleotidase) that confers tolerance to the salts through Agrobacterium tumefaciens co-cultivation. Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly with below 10%, but the 74% tranformation rate were observed at the treatment of phytohormone with 1.0 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryos were initially cultured on MS medium supplemented with 250 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime. Upon development into the cotyledonary stage, these somatic embryos were transferred to on the medium containing 50 mg/l kanamycin and 10 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction with specific primers. The ginseng transformants with well-developed shoots and roots were successfully acclimatized in a greenhouse when they were planted in soil.

• Molecules and Cells
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• v.28 no.4
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• pp.331-339
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• 2009

Capsaicin is a very important secondary metabolite that is unique to Capsicum. Capsaicin biosynthesis is regulated developmentally and environmentally in the placenta of hot pepper. To investigate regulation of capsaicin biosynthesis, the promoter (1,537 bp) of pepper capsaicin synthase (CS) was fused to GUS and introduced into Arabidopsis thaliana (Col-0) via Agrobacterium tumefaciens to produce CSPRO::GUS transgenic plants. The CS was specifically expressed in the placenta tissue of immature green fruit. However, the transgenic Arabidopsis showed ectopic GUS expressions in the leaves, flowers and roots, but not in the stems. The CSPRO activity was relatively high under light conditions and was induced by both heat shock and wounding, as CS transcripts were increased by wounding. Exogenous capsaicin caused strong suppression of the CSPRO activity in transgenic Arabidopsis, as demonstrated by suppression of CS expression in the placenta after capsaicin treatment. Furthermore, the differential expression levels of Kas, Pal and pAmt, which are associated with the capsaicinoid biosynthetic pathway, were also suppressed in the placenta by capsaicin treatment. These results support that capsaicin, a feedback inhibitor, plays a pivotal role in regulating gene expression which is involved in the biosynthesis of capsaicinoids.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.277-283
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• 2009

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for $4{\times}14$ days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with $4mg\;1^{-1}$ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the $R_1$ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

• Journal of Life Science
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• v.11 no.5
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• pp.423-431
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• 2001

Genomic and cDNA clones containing the RAT3 gene involving in Agobacterium-mediated plant transformation were identified using plant DNA flanking the righ border of a T-DNA rescued from the rat3 mutant as hy-bridization probe. Two highly homologous cDNA clones were identified; one (RAT3-1) weakly hybridized with the probe whereas another (RAT3-2) strongly hybridized with the probe. Both Rat3-1 and Rat3-2 proteins contain a putative signal peptide for secretion. The deduced molecular weights of encoded proteins are 15 kDa. The results of genomic DNA blot analysis and DNA sequencing indicated that RAT3-1 and RAT3-2 exist as single copy genes and they were arranged side by side with just 600 bp distance between them. RAT3-1 was disrupted by the integration of T-DNA into the 3 untranslated region in rat3 mutant. A BLAST search showed that both RAT3-1 and RAT3-2 proteins have homology with only the C-terminal region of $\beta$-1,3-glucanase homologues from Triticum aestivum and Arabidopsis thaliana. Thses $\beta$-1,3-glucanase homologues contain an unusually long C-terminal region with no sig-nificant homology to other $\beta$-1,3-glucanase.

• Journal of Plant Biotechnology
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• v.38 no.3
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• pp.209-214
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• 2011

Transgenic lettuce plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with Bcl-2 gene, related to apoptosis. After culture and selection on MS medium a number of kanamycin-resistant plantlets were regenerated. Polymerase chain reaction, Southern blot analysis and Northern blot analysis were used to identify and characterize the transgenic plants with the integrated Bcl-2 gene. Over 100 transgenic plants have been established in soil and flowered in the greenhouse. T1 progeny of 100 transgenic lettuce inbred lines were inoculated with Sclerotinia sclerotiorum. Expression of the Bcl-2 peptide in transgenic lettuce plants provides high levels of field resistance against Sclerotinia sclerotiorum, causal agent of the agronomically important fungal disease of lettuce.

• Journal of Plant Biotechnology
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• v.38 no.3
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• pp.198-202
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• 2011

Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.9-9
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• 2014

Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.

• Korean Journal of Ecology and Environment
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• v.52 no.4
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• pp.394-402
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• 2019

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.213-218
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• 1999

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plants. In addition, it has been suggested that, due to their solubility, they can play an important role in helping plants survive periods of osmotic stress. In order to study the effect of levan synthesis on plant growth, the coding region of the levansucrase gene, which was isolated from Zymomonas mobilis, was introduced into tobacco plants using Agrobacterium tumefaciens-mediated transformation. The presence of the levansucrase gene in transgenic plants was verified by genomic DNA gel blot analysis. RNA gel blot and immunoblot analyses showed an accumulation of the corresponding transcript and protein product of the bacterial levansucrase gene in transgenic plants. Furthermore, a thin layer chromatography analysis revealed that fructans were synthesized and deposited in transgenic tobacco plants. When $T_1$ seeds were germinated and grown under polyethylene glycol-mediated drought stress or cold stress, the transgenic seedlings displayed a substantially higher level of growth than that of untransformed plants. These results suggest that fructans may playa significant role in the tolerance of plants under osmotic stress.