• Applied Biological Chemistry
• /
• v.45 no.4
• /
• pp.203-206
• /
• 2002

${\gamma}-Tocopherol$ methyltransferase (TMT) is an enzyme catalyzing ${\gamma}-tocopherol$ into ${\alpha}-tocopherol$ at the final step of ${\alpha}-tocopherol$ synthesis pathway. Putative TMT cDNA clone specific to Perilla frutescens immature seeds was isolated from cDNA library. The cDNA clone consisted of 1369 bp open reading frame encoding 369 amino acids with a relative Mw of 42 kDa. Results revealed the CDNA has 60% homology to Arabidopsis thaliana TMT, and possesses methyltransferase and S-adenosyl methionine-binding domains, suggesting that cDNA encodes a ${\gamma}-tocopherol$ methyltransferase To characterize the properties of the TMT gene, the cDNA sequences coding for mature TMT were expressed in E. coli and assayed to determine the enzyme activity in vitro.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.6
• /
• pp.435-439
• /
• 2000

Explants of lettuce (Lactuca sativa L.) were cocultured with A. tumefaciens LBA 4404 harboring ${\gamma}$-tocopherol methyltransferase (${\gamma}$-TMT) gene from Arabidopsis thaliana. These explants were transferred to MS medium supplemented with 50 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA and 0.5 mg/L BA. After 4 weeks, kanamycin resistant shoots were obtained from the explants on the selection medium. The putative transgenic shoots were transferred to rooting MS medium supplemented with 50 mg/L kanamycin and 250 mg/L carbenicillin. Stable incorporation of the Arabidopsis ${\gamma}$-TMT cDNA into lettuce genomic DNA was confirmed by PCR and Southern analysis. HPLC analysis showed that $\alpha$- to ${\gamma}$-tocopherol ratio increased over four fold in a transgenic lettuce line indicating successful expression of the transgenic Arabidopsis ${\gamma}$-TMT in lettuce.

• Molecules and Cells
• /
• v.19 no.1
• /
• pp.16-22
• /
• 2005

A cDNA encoding ${\gamma}-tocopherol$ methyltransferase (${\gamma}-TMT$) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce ($T_0$) containing the ${\gamma}-TMT$ transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and ${\gamma}-TMT$ activities. The ratio of ${\alpha}-/{\gamma}-tocopherol$ content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the $T_0$ plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 $T_1$ progenies of the $T_0$ transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and ${\gamma}-TMT$ activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in $T_2$ progenies of $T_1$ plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis ${\gamma}-TMT$ transgene in lettuce results in a higher enzyme activity and the conversion of the ${\gamma}-tocopherol$ pool to ${\alpha}-tocopherol$ in transgenic lettuce.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2000.04a
• /
• pp.420-421
• /
• 2000

• Journal of Plant Biotechnology
• /
• v.35 no.4
• /
• pp.329-335
• /
• 2008

Tocopherols are essential lipophilic antioxidant in human cells, while little is known about its function in plant tissues. To study the impact of composition and content of tocopherols on stress tolerance, tobacco (Nicotiana tabacum) was transformed with a construct containing a cDNA insert encoding $\gamma$-tocopherol methyltransferase ($\gamma$-TMT/VTE4) from perilla under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic tobacco was confirmed by PCR and RT-PCR. The total content and composition of tocopherols in the transgenic lines were similar with wild type controls. However, chlorophyll-a and carotenoid content in the transgenic lines were increased by up to 45% (P<0.01) and 39% (P<0.02), respectively. Also, the over-expression of $\gamma$-TMT increased the salt stress tolerance in tobacco plants. These results demonstrate that over-expression of $\gamma$-TMT gene in tocopherol bio-synthetic pathway can increase salt stress tolerance and contents of chlorophyll-a and carotenoid in transgenic tobacco plants.

• Journal of Plant Biotechnology
• /
• v.7 no.3
• /
• pp.203-209
• /
• 2005

This study was conducted to improve tocopherol (vitamin E) composition in soybean (Glycine max) by introducing a gamma-tocopherol methyl transferase (${\gamma}$-TMT) gene via Agrobacterium tumefaciens-mediated transformation. Immature cotyledon explants were cocultivated with Agrobacterium tumefaciens. Putative transgenic embryos were selected from immature cotyledons on MS medium supplemented with 40 mg/L 2,4-D containing 100 mg/L kanamycin, 500 mg/L carbenicillin and 250 mg/L cefotaxime. Plantlets were developed from somatic embryos, and then transferred to soil. Nineteen regenerated plantlets obtained on the selection medium from 1,460 cotyledons. However, only 9 plantlets were confirmed as transformed plants. Integration of the transgene into the soybean genomic DNA was confirmed by PCR and Southern blot analysis. HPLC analysis showed that the content of ${\alpha}$-tocopherol in transgenic soybean seeds (AT-1) was approximately 4-fold higher than that of non-transgenic plants. Conclusively, we obtained the transgenic soybean having increased ${\alpha}$-tocopherol content by the overexpression of ${\gamma}$-TMT transgene.

• Korean Journal of Medicinal Crop Science
• /
• v.15 no.5
• /
• pp.339-345
• /
• 2007

Field performance and morphological characterization was conducted on seven transgenic lines of Codonopsis lanceolata expressing ${\gamma}-TMT$ gene. The shoots were obtained from leaf explants after co-cultivation with Agrobacterium tume-faciens strain LBA 4404 harboring a binary vector pYBI 121 that carried genes encoding ${\gamma}-Tocopherol$ methyltransferase gene (${\gamma}-TMT$) and a neomycin phosphotransferase II gene (npt II) for kanamycin resistance. The transgenic plants were transferred to a green house for acclimation. Integration of T-DNA into the $T_0\;and\;T_1$ generation of transgenic Codonopsis lanceolata genome was confirmed by the polymerase chain reaction and southern blot analysis. The progenies of transgenic plants showed phenotypic differences within the different lines and with relative to control plants. When grown in field, the transgenic plants in general exhibited increased fertility, significant improvement in the shoot weight, root weight, shoot height and rachis length with relation to the control plants. However, all seven independently derived transgenic lines produced normal flower with respect to its shape, size, color and seeds number at its maturity. Indicating that the addition of a selectable marker gene in the plant genome does not effect on seed germination and agronomic performance of transgenic Codonopsis lanceolata. $T_1$ progenies of these plants were obtained and evaluated together with control plant in a field experiment. Overall, the agronomic performance of $T_1$ progenies of transgenic Codonopsis lanceolata showed superior to that of the seed derived non-transgenic plant. In this study, we report on the morphological variation and agronomic performance of transgenic Codonopsis lanceolata developed by Agrobacterium transformation.

• 한국약용작물학회:학술대회논문집
• /
• 2007.05a
• /
• pp.294-295
• /
• 2007

• 한국약용작물학회:학술대회논문집
• /
• 2007.05a
• /
• pp.296-298
• /
• 2007

• Applied Biological Chemistry
• /
• v.49 no.3
• /
• pp.192-195
• /
• 2006

For the development of a qualitative PCR detection method for genetically modified perilla (Perilla frutescens), perilla species-specific gene, KAS-I (Beta-ketoacyl-ACP synthase I), was selected and validated as suitable for the use as an endogenous reference gene in perilla. Primer specificity was first tested by the means of qualitative PCR analysis. The primer pair Pfru3-F/R amplifying the perilla endogenous gene, KAS-I, gave rise to an amplicon 95 bp. No amplified product was observed when DNA samples from 15 different plants were used as templates. Qualitative PCR detection method was assayed with vitamin E-enriched GM Perilla developed in Korea. For the qualitative PCR detection method, the construct-specific detection primer pairs were constructed. The primer pair TMTO-F/R amplifying the junction region of TMT (${\gamma}$-tocopherol methyltransferase) gene and OCS (Octopine synthase) terminator introduced in GM perilla gave rise to an amplicon 148 bp.