Jae Heung Jeon

Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease

Human β-amyloid (Aβ) is believed to be one of the main components of Alzheimer’s disease, so reduction of Aβ is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Aβ with tandem repeats. Integration of the human Aβ gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Aβ protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Aβ antigen.

Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing d-galacturonic acid reductase gene in response to various abiotic stresses

Upregulation of the antioxidant enzyme system in plants provides protection against various abiotic stresses. Transgenic potato plants overexpressing the strawberry d-galacturonicacidreductase (GalUR) gene with enhanced accumulation of ascorbate (AsA) were used to study the antioxidant system involving the ascorbate–glutathione cycle in order to understand the tolerance mechanism in plants in response to various abiotic stresses under in vitro conditions. Transgenic potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed enhanced activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.1.1.1.6) and enzymes of the ascorbate–glutathione cycle such as ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.8.1.7), as well as increased levels of ascorbate, glutathione (GSH) and proline when compared to untransformed tubers. The increased enzyme activities correlated with the mRNA transcript levels in the stressed transgenic tubers. Significant differences in redox status of AsA and GSH were also observed in stressed transgenic potato tubers that showed increased tolerance to abiotic stresses compared to untransformed tubers. This study suggests that the increased accumulation of AsA could upregulate the antioxidant system which imparts improved tolerance against various abiotic stresses in transgenic tubers compared to untransformed tubers.

Ectopic expression of pepper CaPF1 in potato enhances multiple stresses tolerance and delays initiation of in vitro tuberization

Ethylene-responsive factors (ERFs) are plant-specific transcription factors, many of which have been linked to plant defense responses. However, little is known about the functional significance of ERF genes in potato plants compared to the model plant species Arabidopsis. We show here that overexpression of CaPF1, an ERF/AP2-type pepper transcription factor gene, effectively increased tolerance to freezing, heat, heavy metal, and oxidative stress in potatoes. Interestingly, CaPF1 was involved in tuber formation in potato plants. The time course of microtuber formation was significantly retarded in potato plants that overexpressed CaPF1 compared with wild-type potato plants. Overall, the results of the present study indicate that the pepper transcription factor gene, CaPF1, is involved in promotion of multiple stress tolerance and retardation of in vitro tuberization in potato plants.

Transgenic potato expressing Aβ reduce Aβ burden in Alzheimer’s disease mouse model

Beta amyloid (Aβ) is believed one of the major pathogens of Alzheimers disease (AD), and the reduction of Aβ is considered a primary therapeutic target. Immunization with Aβ can reduce Aβ burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Aβ1–42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Aβ burden in the brain. Thus, Aβ tandem repeats can be expressed in transgenic potato plants to form immunologically functional Aβ, and these potatoes has a potential to be used for the prevention and treatment of AD.

High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice

Plastid transformation has to date been applied to the expression of heterologous genes involved in agronomic traits and to the production of useful recombinant proteins. Here, we report a feasibility study for producing the human β-site APP cleaving enzyme (BACE) via transformation of tobacco chloroplasts. Stable integration of human BACE into the plastome was confirmed by PCR. Genomic Southern blot analysis detected the presence of the tobacco aadA and human BACE genes between trnI and trnA in the plastome. Northern blot analysis revealed that the aadA and BACE genes were both properly transcribed into a dicistronic transcriptional unit. Human BACE protein expression in transplastomic tobacco was determined by western blot analysis. ELISA analysis revealed that, based on a dilution series of E. coli-derived BACE as a standard, transplastomic lines accumulated BACE to levels of 2.0% of total soluble proteins. When mice were gavaged with the transplastomic tobacco extracts, they showed an immune response against the BACE antigen. The successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease.

Polyamine Biosynthesis Regulated by StARD Expression Plays an Important Role in Potato Wound Periderm Formation

An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.

Effects of rindite on breaking dormancy of potato microtubers

Potato microtubers were treated with rindite to investigate the effect on dormancy breaking. Postharvest application of rindite by fumigation with 2 ml rindite for 48 hr or 4ml for 24 hr significantly reduced the dormancy period of potato microtubers using a 32 x 15 x 17 cm tightly sealed plastic box. Approximately 2 weeks after the treatments microtubers of all cultivars, Atlantic, Superior, Lemhi Russet, Red Dale and Kennebec started to sprout. The efficiency of the treatments were the greatest for the cv. Lemhi Russet, intermediate for cv. Superior and least for cv. Red Dale. In all cultivars of potato microtubers, more decay was observed the earlier rindite treatment occurred after harvesting, therefore potato microtubers should be treated with rindite at least 2 weeks after harvest when the skin of microtubers is mature. The data indicates that the dormancy of potato microtubers with well-matured skin can be effectively broken with an optimum treatment of rindite.

Antibody Responses in Mice Stimulated by Various Doses of the Potato-Derived Major Surface Antigen of Hepatitis B Virus

ABSTRACT The ability of potato-derived major surface antigen of hepatitis B virus (P-HBsAg) to elicit antibody responses to different dosages of P-HBsAg ranging from 0.02 to 30 μg administered orally in mice was examined. All immunized groups produced specific serum IgG and fecal IgA antibodies against P-HBsAg, even at low levels (<5 μg), after administration of a 0.5-μg yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) booster.

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8xa0mgu2009·u2009g−1 leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.