• The Plant Pathology Journal
• /
• v.22 no.4
• /
• pp.360-363
• /
• 2006

Efficacy of plant growth promoting rhizobacteria(PGPR) Bacillus vallismortis strain EXTN-1 has been proved in eliciting induced systemic resistance(ISR) in several crops. The present paper described the beneficial effects of EXTN-1 in potato as increase in yield and chlorophyll content, and plant protection against Potato Virus Y and X(PVY & PVX). EXTN-1 induced systemic resistance to the plants resulting in significant disease suppression in the field. Also the plants under treatment with EXTN-1 had higher chlorophyll content. The bacterized plants had significantly higher yields over the untreated control plants. The strain induced activation of defense genes, PR-1a and PDF 1.2 in transgenic tobacco model, which indicated the possible role of both SA, and JA pathways in EXTN-1 mediated plant protection against crop diseases.

• Journal of Plant Biotechnology
• /
• v.40 no.3
• /
• pp.169-177
• /
• 2013

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus ${\rightarrow}$ tobacco E2 signal peptide ${\rightarrow}$ B-peptide (1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-peptide (1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.

• Journal of Plant Biotechnology
• /
• v.35 no.1
• /
• pp.87-94
• /
• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Journal of Plant Biotechnology
• /
• v.37 no.3
• /
• pp.250-261
• /
• 2010

The expression of vaccine antigens in transgenic plants has the potential to provide a convenient, stable, safe approach for oral vaccination alternative to traditional parenteral vaccines. Over the past two decades, many different vaccine antigens expressed via the plant nuclear genome have elicited appropriate immunoglobulin responses and have conferred protection upon oral delivery. Up to date, efforts to produce antigen proteins in plants have focused on potato, tobacco, tomato, banana, and seed (maize, rice, soybean, etc). The choice of promoters affects transgene transcription, resulting in changes not only in concentration, but also in the stage tissue and cell specificity of its expression. Inclusion of mucosal adjuvants during immunization with the vaccine antigen has been an important step towards the success of plant-derived vaccines. In animal and Phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Future areas of research should further characterize the induction of the mucosal immune response and appropriate dosage for delivery system of animal and human vaccines. This article reviews the current status of development in the area of the use of plant for the development of oral vaccines.

• Proceedings of the Botanical Society of Korea Conference
• /
• 1999.07a
• /
• pp.79-84
• /
• 1999

Oxidative stress is one of the major environmental stresses to plants. Reactive oxygen species (ROS) generated during metabolic processes damage cellular functions and consequently lead to cell death. Fortunately plants have in vivo defense system by which the ROS is scavenged by enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). In attempts to understand the protection mechanism of plant against oxidative stress, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plansts thet expressed both SOD and APX in chloroplast using Agrobacterum-mediated transformation and evaluated their protection capabilities against methyl viologen (MV, paraquat) -mediated oxidative damage. Three double transformants (CAI, CA2, and CA3) expressed the chimeric CuZnSOD and chimeric APX in chloroplast, and one transformant (AM) expressed the chimeric APX and chimeric MnSOD in chloroplast. In addition, we obtained three lines of transformants (C/Al, C/A2, and A/C) that expressed the APX and SOD than control plants, and more resistant to oxidative stress caused by MV. TRansformants (C/A and A/C) overexpressing MnSOD, CuZnSOD and APX at the same time showed the highest resistance to MV-mediated oxidative stress among the transformants.

• Journal of Plant Biotechnology
• /
• v.29 no.4
• /
• pp.229-233
• /
• 2002

Ferritin is ubiquitous in bacteria, animals and plants. Ferritin is thought to play two main roles in living cells to provide iron for the synthesis of iron protein such as ferretoxin and cytochromes and to prevent damage from radicals produced by iron/dioxygen interaction. To enhance the resistance of potato to Erwinia carotovora, the soybean ferritin gene was introduced into the potato either under CaMV 35S or hsr203J promoter. Potato transgenic plants were screened by PCR analysis using specific primers to the ferritin gene. Expression of ferritin gene under CaMV 35S and hsr203J promoter in potato transgenic plants was confirmed by northern blot analysis. hsr203J promoter known to pathogen inducible in tobacco drives the induction upon Phytophthora infestan in potato and the transcript level of ferritin gene was extremely high after 24 hours post inoculation. One of transformants under CaMV 35S promoter was increased 2.5 fold than untransformant. Each one of transgenic potato containing gene promoter CaMV 35S and hsr203J-ferrtin fusion exhibited tolerance against potato soft rot.

• Journal of Life Science
• /
• v.15 no.2 s.69
• /
• pp.176-181
• /
• 2005

To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to $\beta-glucuronidase$ (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5'-flanking region of RCaM gene regulates wound-inducible expression.

• Korean Journal of Plant Tissue Culture
• /
• v.22 no.4
• /
• pp.201-205
• /
• 1995

The synthetic oligomers called nos right palindrome (RP) element and left palindrome (LP) element were inserted into nos.minimal promoter nos 5'-101 deletion mutant The activity of nos promoter was measured by studying the expression pattern of gene fusion between nos promoter and reporter genes such as chloramphenicol acetyltransferase and $\beta$-glucuconidase. Analysis of transgenic tobacco plane carrying transgene showed that the activity of nos minimal promoter activity was recovered by insertion of synthetic nos RP element. Nos RP element insertion of nos minimal promoter was induced by auxin, dithiothreitol, salicylic acid and methyl jasmonate.

• Horticultural Science & Technology
• /
• v.17 no.4
• /
• pp.473-475
• /
• 1999

This study was carried out to restore the fertility by suppression of male sterility-induced gene using an antisense construct. Tobacco (cv. Petit Havana SR1) was transformed with the binary vector containing a GBAN215-6 promoter, an antisense diphtheria toxin (DTx-A) gene (pKDA215b) and a hygromycin resistant gene. Seventy-six confirmed transgenic plants regenerated from leaf disks were designated as the $R_0$ generation and selfed to produce the $R_1$ generation. From the inheritance study, five $R_1$ lines with multiple copies of the antisense construct were selected and selfed to identify homozygosity for the antisense construct. In order to restore fertility and finally to select restore lines, five $R_2$ lines with multiple copies of the antisense construct were crossed with male sterile plants. From these crosses, three different phenotypes have been observed: completely restored, partially restored, and not restored pollens, and otherwise tobacco plants were phenotypically same as normal plants. These plants were scored for the degree of restoration and selected for further study.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.13 no.3
• /
• pp.177-183
• /
• 1993

This study was conducted to obtain the transformed tobacco plants with S. cerevisiae Acid phosphatase gene(PH05) using Agrobacterium tumefaciens and th confirm plant transformation and gene expression. the results obtained were summarized as follows: APase activity of Saccharomyces cereviase NA 87-11A was remarkably showed up as deep red color when assayed by Tohe and Oshima(1974). PH05 fragment, Apase gene, was obtained from pVC727G and the graphically estimated size was about 1.5kb by agarose gel electrophoresis. The sequencing results of 5'end and 3'end of PH05 using dideoxy chain termination method were coinsided with the full length nucleotide already. pBKJ I vector was constructed by isolation of PH05 fragment from pVC727-1 and pBKSI-1 digesred with Sma I and Xba I. Isolated plasmid from transformed A. tumefaciens with constructed pBKJ I when it was electrophoresed with agarose gel. The dosc of tobacco leaf was cocultivated 재소 transformed Agronacterium tumefaciens. Transformed shoots were selected on kanamtcin-containing MS-n/B medium and they were regenerated. The transgenic tobacco plants were elucidated by isolation of genomic DNA and genomic southern hybridization using ${\alpha}-^{32}P$ labelled PH05 fragments. The PH05 in transformed tobacco plants was expressed in leaf, stem and root, and its APase activity was estimated as deep red color by Tohe method.