Kyoung-Sil Yang

Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweetpotato plants.

Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of an oxidative stress–inducible peroxidase (SWPA2) promoter (referred to as SN plants) were developed and evaluated for enhanced tolerance of SN plants under various abiotic stress conditions. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant’s tolerance to MV. Interestingly, we observed that antioxidant enzyme activities such as peroxidase, ascorbate peroxidase, and catalase increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to cold, high salinity, and drought stresses by an increase in the activity of H2O2 scavenging enzymes. These results indicate that overexpression of AtNDPK2 in sweetpotato might efficiently modulate oxidative stress from various environmental stresses.

Silencing of NbBTF3 results in developmental defects and disturbed gene expression in chloroplasts and mitochondria of higher plants

BTF3 (βNAC) was originally isolated as a general transcription factor required for RNA polymerase II-dependent transcription, and later found to be a β-subunit of nascent-polypeptide-associated complex that has been implicated in regulating protein localization during translation. In this study, virus-induced gene silencing of NbBTF3 encoding a Nicotiana benthamiana homolog of human BTF3 caused leaf yellowing and abnormal leaf morphology without altering the overall growth of the plant. The NbBTF3 gene is constitutively expressed and the NbBTF3–GFP fusion protein is primarily targeted to the nucleus. At the cellular level, downregulation of NbBTF3 expression reduced the chloroplast sizes and chlorophyll contents. The affected cells produced excessive amounts of reactive oxygen species. Furthermore, the transcript level of various plastid- and mitochondria-encoded genes was severely reduced in the NbBTF3-depleted leaf cells. These findings indicate that depletion of NbBTF3 activity preferentially affected development and/or physiology of chloroplasts and mitochondria in plants, possibly by hampering efficient translocation of the nascent organellar proteins into the organelles.

High-yield production of functional human lactoferrin in transgenic cell cultures of siberian ginseng (Acanthopanax senticosus)

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducibleSWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

Agrobacterium- mediated Genetic Transformation and Plant Regeneration of Sweetpotato (Ipomoea batatas)

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron -glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

Selection of oxidative stress-tolerant sweetpotato cultivars for cultivation on marginal lands

Abstract Oxidative stress derived from excess reactive oxygen species (ROS) is a major damaging factor for plants exposed to environmental stresses. Sweetpotato [ Ipomoea batatas (L.) Lam] has a relatively broad adaptability to harsh environmental conditions compared to other staple crops. In this study, to select stress-tolerant sweetpotato cultivars for sources of molecular breeding on marginal lands, we eval-uated the ion leakage values in 10 different cultivars after treatment of methyl viologen (MV), an ROS-generating non- selective herbicide, to leaf discs. DPPH radical scavenging activity and the contents of total phenolics were also inves-tigated. The ion leakage of each cultivar showed a diverse value, which is well correlated with DPPH radical scavenging activity of each cultivar. DPPH radical scavenging activity also showed a high corelation with the contents of total phenolic contents. Three cultivars of Yanshu 8, Shinhwangmi and Shinzami showed high antioxidant activity. Our results suggest that a simple and efficient DPPH radical scavenging activity would be a suitable method to select potential cul-tivars with enhanced tolerance to multiple environmental stress.

Current status on metabolic engineering of starch in sweetpotato

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Progress in understanding of starch biosynthesis, and the isolation of many genes involved in this process have enabled the genetic modification of crops in a rational manner to produce novel starches with improved functionality. Starch is composed of two glucose polymers, amylose and amylopectin. The amylose and amy- lopectin ratio in starch affects its physical and physic- ochemical properties. Alteration in starch structure can be achieved by modifying genes encoding the enzymes responsible for starch biosynthesis and starch hydrolysis. Here, we describe recent findings concerning the starch modification in sweetpotato. Sweetpotato (Ipomoea batatas (L.) Lam) ranks seventh in annual production among food crops in the world as an important starch source. To develop transgenic sweetpotato plants with modifying starch com- position, we constructed transformation vectors overex- pressing granule bound starch synthase I and inhibiting amylopectin synthesis genes such as starch branching enzyme and isoamylase under the control of 35S promoter, respectively. Transformation of sweetpotato (cv. Yulmi) is

Transgenic tobacco culture cells expressing spike protein gene of porcine epidemic diarrhea virus

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.