• Journal of Plant Biology
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• v.37 no.3
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• pp.371-380
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• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.37-43
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• 1998

The highest transformation frequency was observed when cotyledonary and hypocotyl explants of flowering cabbage (Brassica oleracea var. acephala DC) 'Eunbae' were cultured on shoot induction medium without kanamycin for 1 day, then cocultured with Agrobacterium tumefaciens LBA4404;;pGA1036 harboring tomato inhibitor II promoter and $\beta$-glucuronidae (GUS) fusion gene for 3 days. These explants were transferred to MS medium containing 20 mg/L kanamycin, 500 mg/L carbenicillin, and 1 mg/L BA. The explants were subsequently subcultured every 2 weeks. Incorporation of the GUS gene into flowering cabbage was confirmed by PCR analysis of DNA. Southern blot analysis showed that ECL-labeled GUS gene was hybridized to the expected amplified genomic DNA fragment of about 366 bp from transgenic flowering cabbage. Histochemical analysis based on the enzymatic activity of the GUS protein indicated that PI-II promoter activity was sysmatically associated with vascular tissue in wonded as well as in non-wounded leaves, petioles and stems, but not in roots. Partial wounding with razor blade showed not systemic induction but partial induction.

• Molecules and Cells
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• v.26 no.5
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• pp.474-480
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• 2008

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.

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

• The Plant Pathology Journal
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• v.23 no.2
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• pp.76-89
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• 2007

CaCDPK4, a full-length cDNA clone encoding Capsicum annuum calcium-dependent protein kinase 4, was isolated from chili pepper (Capsicum annuum L.). Deduced amino acid sequence of CaCDPK4 shares the highest homology with tobacco NpCDPK8 and chickpea CaCDPK2 with 79% identity. Genomic blot analyses revealed that CaCDPK4 is present as a single copy in pepper genome, but it belongs to a multigene family. CaCDPK4 was highly induced when pepper plants were inoculated with an incompatible bacterial pathogen. Induced levels of CaCDPK4 transcripts were also detected in pepper leaves by the treatment of ethephon, an ethylene-inducing agent, and high-salt stress condition. The bacterial-expressed GST-CaCDPK4 protein showed to retain the autophosphorylation activity in vitro. GUS expression driven by CaCDPK4 promoter was examined in transgenic Arabidopsis containing transcriptional fusion of CaCDPK4 promoter. GUS expression under CaCDPK4 promoter was strong in the root and veins of the seedlings. GW (-1965) and D3 (-1377) promoters conferred on GUS expression in response to inoculation of an incompatible bacterial pathogen, but D4-GUS (-913) and DS-GUS (-833) did not. Taken together, our results suggest that CaCDPK4 can be implicated on signal transduction pathway of defense response against an incompatible bacterial pathogen in pepper.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.315-318
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• 1994

To obtain transformed plants, we cocultured cotyledonary explants of Codonopsis lanceolata with Agrobacterium tumefaciens LBA4404, a disamed strain harboring a binary vector pBI121 carrying the CaMV35S promoter-$\beta$-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker in MS liquid medium with 1mg/L BA. After 48 h of culture, explants were transferred onto MS solid medium with Img/L BA, 250mg/L carbenicillin, and 100mg/L kanamycin sulfate and cultured in the dark. Numerous adventitious buds formed on the cut edges of the explants after 2 weeks of culture. When subjected to GUS histochemical assay buds showed a positive response at a frequency of 15%. Explants formed adventitious shoot at a frequency of 56.7%, after 6 weeks of culture. Upon transfer onto the basal medium, most of the shoots were rooted and subsequently the regenerants were transplanted to potting soil. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants.

• KSBB Journal
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• v.16 no.5
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• pp.480-485
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• 2001

Genetic transformation in dandelion(Taraxacum mongolicum Hand). was studied. We used for transformation by Agrobacterium tumefaciens strian LBA4404 harboring a binary vector pBI121 carrying the CaMV 35S promoter-GUS gene fusion used as a reporter gene and NOS promoter-NPTII gene as a positive selection marker. To obtain transformed plants, leaf explants of dandelion were cocultured with Agrobacterium tumefaciens LBA4404 for 10 mins, then transferred to MS medium containing 1 $\mu$M IAA, 1$\mu$M BA, 100$\mu$g/ML carbenicillin and 50 $\mu$g/ML kanarmycin sulfate. After two weeks of subculture of the explants, Kanamycin-resistant shoots were formed on explants survived. When subjected to GUS histochemical assay, all of the regenerants showed the GUS-positive responses. Plantlets were be be transformed to soil for further growth.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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• pp.434-439
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• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.356-363
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• 2015

In order to study genetic engineering in trees, the characterization of genes and promoters from trees is necessary. We isolated the promoter region (867 bp) of Pagns-LTP from poplar (P. alba ${\times}$ P. glandulosa) and characterized its activity in transgenic poplar plants using a ${\beta}$-glucuronidase (GUS) reporter gene. High-level expression of the Pagns-LTP transcript was found in poplar roots, while comparatively low-level expression was found in the young leaves. Pagns-LTP mRNA was not detected in other poplar tissues. Additionally, transgenic poplar plants that contained a Pagns-LTP promoter fused to a GUS reporter gene, displayed tissue-specific GUS enzyme activity localized in root tissue. In silico analysis of the Pagns-LTP promoter sequence reveals the presence of several cis-regulatory elements responsive to phytohormones, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrate that the Pagns-LTP promoter has tissue-specific expression activity in poplar roots and leaves that may be involved in organ development and plant resistance to various stresses. Therefore, we anticipate that the Pagns-LTP promoter would be a useful tool to genetically optimize woody plants for functional genomics.

• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.13-18
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• 2003

This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.