• Journal of Plant Biotechnology
• /
• v.4 no.4
• /
• pp.143-150
• /
• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• Plant Biotechnology Reports
• /
• v.3 no.4
• /
• pp.325-331
• /
• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• BMB Reports
• /
• v.41 no.11
• /
• pp.771-777
• /
• 2008

Calmodulin (CaM) proteins, members of the EF-hand family of $Ca^{2+}$-binding proteins, represent important relays in plant calcium signals. Here, OsCam1-1 was isolated by PCR amplification from the rice genome. The gene contains an ORF of 450 base pairs with a single intron at the same position found in other plant Cam genes. A promoter region with a TATA box at position-26 was predicted and fused to a gus reporter gene, and this construct was used to produce transgenic rice by Agrobacterium-mediated transformation. GUS activity was observed in all organs examined and throughout tissues in cross-sections, but activity was strongest in the vascular bundles of leaves and the vascular cylinders of roots. To examine the properties of OsCaM1-1, the encoding cDNA was expressed in Escherichia coli. The electrophoretic mobility shift when incubated with $Ca^{2+}$ indicates that recombinant OsCaM1-1 is a functional $Ca^{2+}$-binding protein. In addition, OsCaM1-1 bound the CaMKII target peptide confirming its likely functionality as a calmodulin.

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

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.31 no.1
• /
• pp.1-8
• /
• 2011

Molecular techniques such as genetic transformation are powerful tools that can be used for the genetic modification of warm-season grasses. The P. meyerianum with high regeneration ability was used for establishing an Agrobacterium-mediated transformation system. We investigated various factors affecting Agrobacterium infection by examining GUS gene expression of pCAMBIA1304 vector. Among various concentration of acetosyringone and betaine tested for inoculation and co-cultivation, 10 mg/L acetosyringone and 60 mg/L betaine resulted in the highest transformation frequency in terms of GUS expression. The calli of 4 species of Panicum spp. with excellent tissue culture response were inoculated with Agrobacterium under the optimal infection conditions. The high activity of GUS gene was observed in all species and hygromycin-resistant calli expressing GFP were obtained in P. meyerianum, P. longijubatum, P. stapfianum and guineagrass Noh-PL1. Co-cultivated calli were transferred onto the selection medium containing hygromycin, and the hygromycin resistant calli were selected after 3 months. Hygromycin-resistant plantlets were then successfully regenerated from the calli and grown in a greenhouse. We confirmed stable insertion of hpt gene among the hygromycin-resistant plantlets of P. meyerianum by PCR analysis.

• Journal of Plant Biotechnology
• /
• v.33 no.1
• /
• pp.49-56
• /
• 2006

Gerber (Gerbera hybrida) is a valuable ornamental species grown as a potted plant and cut flowers. However, genetic variability within the gerbera genus is very limited. So it is absolutely needed to introduce and widen genetic resources into gerbera lines by genetic transformation. For the purpose, 18 Korean gerbera lines were screened to establish Agrobacterium-mediated genetic transformation procedure. In an experiment to select Korean gerbera lines which are amenable to Agrobacterium-inoculation, 12 lines turned out to be positive in Agrobacterium-inoculation. More callus were produced from BA 2ppm, Zeatin 2ppm, IAA 0.2ppm in pre-culture and regeneration medium (2X media) but there was no difference in the frequency of GUS expression rate. In another experiment to find out optimal condition for highly efficient Agrobacterium-inoculation, petiole and leaf explants have been treated with four different pre-culture periods, two different co-culture periods and two different Agrobacterium strains. As a result, high GUS expression has been showed from petiole and leaf explants treated no pre-culture period with LBA4404 Agrobacterium tumerfaciens, 5 day co-culture period and dipping treatment.

• Journal of Plant Biotechnology
• /
• v.2 no.2
• /
• pp.61-71
• /
• 2000

By screening a cDNA library of auxin-treated mung bean (Vigna radiata L.) hypocotyls, we have isolated two full-length cDNA clones, pVR-ACS6 and pVR-ACS7, for 1-aminocyclopropane-1-carboxylate (ACC) synthase, the rate-limiting enzyme in the ethylene biosynthetic pathway. While PVR-ACS6 corresponds to the previously identified PCR fragment pMBA1, pVR-ACS7 is a new cDNA clone. A comparison of deduced amino acid sequences among auxin-induced ACC synthases reveal that these enzymes share a high degree of homology (65-75%) to VR-ACS6 and VR-ACS7 polypeptides, but only about 50% to VR-ACS1 polypeptide. ACS6 and ACS7 are specifically induced by auxin, while ACS1 is induced by cycloheximide, and to lesser extent by excision and auxin treatment. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, whereas W-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the $\beta$-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tobacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.5
• /
• pp.694-699
• /
• 2000

Serratia marcescens IRBG500 and Herbaspirillum seropedicae Z67 grow endophytically in rice. The ability of these bacteria to colonize rice grown under increased nutrient availability was assessed in variety IR72 using strains marked with transposon-based gusA. The endophytic colonization was monitored via bacterial enumeration and histochemical visualization of GUS expression of bacteria in plant tissues. Rhizoplane and endophytic colonization by both bacteria was significantly inhibited in the rice plants grown in the presence of 10 mM $NH_4Cl$. In contrast, the addition of 10 mM $KNO_3$ showed no adverse effect on colonization. Increasing the concentration of $Ca^{2+}$ to 5 mM significantly reduced endophytic colonization by both bacterial strains, whereas the addition of 0.5 mM $Fe^{2+}$ substantially lowered the colonization of roots by S. marcescens IRBG500 but showed no effect on colonization by H. seropedicae Z67. Taken together, these finding suggest that, like in legume-rhizobial symbiosis as well as plant-pathogen interactions, nutrient status, particularly $NH_4^+$ and $Ca^{2+}$ concentrations in the surrounding medium, plays an important role in the regulation of endophytic infection and colonization processes in rice.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.34 no.3
• /
• pp.187-192
• /
• 2014

Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world. It has been demanded to establish the efficient transformation system in commercial varieties of alfalfa for forage molecular breeding and production of varieties possessing new characteristics. To approach this, genetic transformation techniques have been developed and modified. This work was performed to establish conditions for effective transformation of commercial alfalfa cultivars, Xinjiang Daye, ABT405, Vernal, Wintergreen and Alfagraze. GUS gene was used as a transgene and cotyledon and hypocotyl as a source of explants. Transformation efficiencies differed from 0 to 7.9% among alfalfa cultivars. Highest transformation efficiencies were observed in the cultivar Xinjiang Daye. The integration and expression of the transgenes in the transformed alfalfa plants was confirmed by polymerase chain reaction (PCR) and histochemical GUS assay. These data demonstrate highly efficient Agrobacterium transformation of diverse alfalfa cultivars Xinjiang Daye, which enables routine production of transgenic alfalfa plants.

• Mycobiology
• /
• v.32 no.1
• /
• pp.1-5
• /
• 2004

The present study was carried out to investigate morphological characteristics of pseudosclerotia of Grifola umbellata formed by artificial cultures. Isolate G. umbellata DUM GUS-01 was obtained from sclerotium cultivated in field. The fungal isolate was cultured on PDYM broth, PDYMA(potato dextrose yeast malt agar) and oak sawdust media at $20^{\circ}C$ under the dark condition. G. umbellata DUM GUS-01 showed a volumetric increment of fungal lumps rather than mycelial growth. Particularly, G. umbellata DUM GUS-01 produced a large amount of melanin pigments in all culture treatments. The color of the fungal mass has been changed into grey gradually, and then formed melanized rind-like structure on its superficial part. The fungal structures which were covered with melanized rind-like layer were named as pseudosclerotia of G. umbellata. The pseudosclerotia of G. umbellata DUM GUS-01 formed a new white mycelial mass, which was swollen out of the melanized rind structure for its volumetric increment. When the pseudosclerotia were sectioned, their structure was discriminated from two structures such as a melanized rind-like structure layer formed by aggregation of aged mycelia and a white mycelial mass with high density. As results of scanning electron microscopic examination, the pseudosclerotia of G. umbellata DUM GUS-01 which were formed in in vitro conditions were similar to the sclerotia of G. umbellata cultivated in natural conditions except for the crystals formed in medula layer of natural sclerotia. Although size, solidity of rind structure and mycelial compactness of pseudosclerotia were more poor than those of natural sclerotia, the morphological structure and growth pattern of pseudosclerotia were very similar to those of natural sclerotia. Therefore, it is probable to induce pseudosclerotia to sclerotia of G. umbellata in in vitro conditions. Consequently, it seems that the induced pseudosclerotia can be used as inoculum sources to substitute natural sclerotia in field cultivation.