Vinca alkaloids from plant Vinca minor have been investigated for their effects of tyrosinase inhibition, stimulation of ROS generation and increasement of cell migration activity. The methanolic crude extract and the water-soluble fraction exhibited IC50 value of 3.1 mg/mL and 2.1 mg/mL. Vinca minor extract treatment significantly increased ROS levels in HaCaT cells, in a concentration-dependent manner. Treatments of Vinca minor extract led to increase wound closure when compared with non-treatment. Low dose (0.1% or 0.3%) of extracts have not significantly affected, compared with that in controls. By contrast, 0.5% extract have dramatic effect on wound healing activity of keratinocytes. Effects of Vinca minor extract in a filter-based cell mobility assay appear similar to that of wound closure assay, which suggests that the Vinca minor extract have wound healing effects on skin.

Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.

A large number of transgenic crop varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins have been commercialized in 13 countries since 1996. Although the use of these insect-resistant Bt crops can increase crop quality and yields, concerns remain about the potential negative effects of such crops on ecosystems. Transgenic soybean containing cry1Ac gene have been developed to control Lepidopteran pests of soybean and we aimed to investigate whether this soybean could affect non-target arthropods, which play a major role in ecological functions in agricultural ecosystems. In the present study, we first measured the levels of Cry1Ac protein in Bt soybean at different growth stages of soybean and then we compared the community structure of arthropods occurred in fields of transgenic and wild-type soybean. The levels of Cry1Ac protein in transgenic soybean leaves ranged from 252.9 to 604.5 μg g-1 DW. Multivariate analyses (PerMANOVA and NMDS) showed that the composition of the non-target arthropod community was affected by sampling date but not by soybean genotype. These results suggest that transgenic soybean expressing Cry1Ac protein may not adversely affect such non-target arthropod communities.

지진하중파 유사한 반 복 하증에 의한 철금 콘크리 프 부재의 설제 거동을 재생키 위한 해석적인 이력 모덴블 제시하였다. 특 히 RC 샤재의 동적거동의 증요한 현상뜰언 강성서하， 강도저하 그리고 전단 영향등의 해석 모 덴 올 소개하였으며 제안된 이력모덴의 정 확성 및 사용성 동 의 평가플 위하여 설험결과 의 하 중 변위 팍선윌 과 비 j니 분 석 하였다

This study aimed to produce fermented soy-powder milk (FSPM) with Lactobacillus plantarum P1201 and to evaluate its anti-obesity activity. Isoflavone and conjugated linoleic acid (CLA) of unfermented soy-powder milk (UFSPM) and FSPM and were analyzed via high-pressure liquid chromatography (HPLC) and gas chromatography (GC). Their inhibitory activities against α-glucosidase, α-amylase, and pancreatic lipase were assayed. Their anti-obesity activities were evaluated on the basis of their inhibitory effects on adipocyte differentiation in 3T3-L1 cells, and the expression of mRNAs associated with adipogenesis and lipid metabolism were analyzed via real time-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR). FSPM with L. plantarum P1201 increased the isoflavone aglycones (daidzein, glycitein, and genistein) content and produced CLA in soy-powder milk (SPM), both of which possessed bio-activity. Both UFSPM and FSPM showed dose-dependent inhibitory activity for α-glucosidase, α-amylase, and pancreatic lipase. FSPM, but not UFSPM, suppressed adipogenesis in 3T3-L1 cells and reduced their triglyceride content by 23.1% after treatment with 1,000 μg/mL of FSPM, compared with the control group. The anti-obesity effect of FSPM can be attributed to CLA and isoflavone aglycones, which targeted CCAAT/enhancer binding protein α (C/EBP-α) and down-regulated lipoprotein lipase (LPL), adiponectin, adipocyte fatty acid-binding protein (aP2), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) mRNA. Furthermore, FSPM enhanced the inhibitory activity of glucosidase and pancreatic enzymes and anti-obesity activity. Further studies are required to investigate whether the anti-obesity effect of FSPM persists in an in vivo mouse model of diet-induced obesity.

High yield is the most important trait in various agricultural characteristics. Many approaches to improve yield have been tried in conventional agricultural practice and recently biotechnological tools employed for same goal. Genetic transformation of key genes to increase yield is one way to overcome current limitation in the field. We are producing transgenic soybean plants through high efficient transformation method by introducing all gene member with AT-hook binding domain, hoping to obtain manageable delay of senescence. Many transgenic soybean plants are growing in greenhouse and GMO field, and will be evaluated their senescence and any association with yield increase.

AtRabG3b and CaMsrB2 genes incorporated into pPZP vetor were transformed to Korean soybean cultivar Kwangan using highly efficient transformation system. AtRabG3b gene plays a positive role in xylem development in Arabidopsis and 64 transgenic plants were produced. CaMsrB2 gene is known to confer drought tolerance in rice and 63 transgenic plants were produced. As a result of PPT leaf painting assay, about 20% of transformation efficiency was observed from 2 times of inoculation. These transgenic plants were confirmed for gene introduction using PCR. Currently, the copy number and the gene expression is investigating using qRT-PCR and RT-PCR. Moreover, 62 lines and 53 lines of T1 seeds from AtRabG3b and CaMsrB2, respectively, were sown in GMO field.

ORE7 gene incorporated into 3 different promoters including pCKLSL-35S, pCKLSL-TP and pCSENIF was transformed to Korean soybean variety Kwangan using highly efficient soybean transformation system. The gene is known to exhibit a delayed leaf senescence phenotype in Arabidopsis. Fourteen, Fifteen and nine transgenic plants were produced from pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7, respectively. Moreover, transgenic plants were confirmed for gene introduction and their expression using PCR, qRT-PCR and RT-PCR. To identify the transgene insertion events, the analysis of flanking sequence was determined. As a results, T-DNA was integrated intergenically in transgenic line 1 of pCKLSL-35S::ORE7 and line 1 of pCSENIF::ORE7. Currently, flanking sequence analysis with pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7 is carrying out to investigate the stable T-DNA insertions.

Insect resistant genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korean soybean variety Kwangan was transformed with Insect resistant genes. These genes were transformed into Kwangan using highly efficient soybean transformation system. Transgenic plants harboring Insect resistant genes were confirmed for gene introduction and their expression using PCR, real-time PCR and RT-PCR. The confirmation of stable gene introduction with Insect resistant genes was also performing by Southern blot analysis. In addition, Flanking sequence analysis and agronomic characters were also investigated

Soybean mosaic virus (SMV), a member of Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing SMV-CP and HC-Pro gene by RNA interference (RNAi) method via Agrobacterium-mediated transformation. Transgenic plants were infected with SMV strain G5 and investigated the viral response. As a result, two lines (3 and 4) of SMV-CP(RNAi) transgenic plants and three lines (2, 5 and 6) of HC-Pro(RNAi) transgenic plants showed viral resistance. In genomic Southern blot analysis, most of lines contained at least one T-DNA insertion in both SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. Subsequent investigation confirmed that no viral CP and HC-Pro gene expression was detected in two SMV-resistant lines of SMV-CP(RNAi) and three lines of HC-Pro(RNAi) transgenic plants, respectively. On the other hand, non-transgenic plants and other lines showed viral RNA expression. Viral symptoms affected seed morphology, and clean seeds were harvested from SMV-resistant line of SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. In addition, strong viral gene expression was detected from seeds of SMV-susceptible non-transgenic plants and SMV-susceptible transgenic lines. When compared the viral resistance between SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants, soybean transgenic plants with the HC-Pro gene using RNAi strategy showed much stronger and higher frequency of viral resistance.

UGT72E3/2 gene encodes UDP-glycosyltransferase shown to glucosylate several phenylpropanoids such as syringin and coniferin. Syringin has effect of anti-stress and anti-fatigue. Korean soybean variety Kwangan was transformed with UGT72E3/2 gene. This gene was transformed into Kwangan using highly efficient soybean transformation system. This study used two promoters, beta-conglycinin promoter for seed-specific expression and 35s promoter for total expression. Transgenic plants were confirmed for gene introduction and their expression using PCR and RT-PCR. The analysis of syringin in transgenic plants was performed using HPLC. Currently, the confirmation of stable gene introduction with UGT72E3/2 gene is also performing by Southern blot analysis.

Soybean mosaic virus (SMV), a member of Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing SMV-CP and HC-Pro gene by RNA interference (RNAi) method via Agrobacterium-mediated transformation. Transgenic plants were infected with SMV strain G5 and investigated the viral response. As a result, two lines (3 and 4) of SMV-CP(RNAi) transgenic plants and three lines (2, 5 and 6) of HC-Pro(RNAi) transgenic plants showed viral resistance. In genomic Southern blot analysis, most of lines contained at least one T-DNA insertion in both SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. Subsequent investigation confirmed that no viral CP and HC-Pro gene expression was detected in two SMV-resistant lines of SMV-CP(RNAi) and three lines of HC-Pro(RNAi) transgenic plants, respectively. On the other hand, non-transgenic plants and other lines showed viral RNA expression. Viral symptoms affected seed morphology, and clean seeds were harvested from SMV-resistant line of SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. In addition, strong viral gene expression was detected from seeds of SMV-susceptible non-transgenic plants and SMV-susceptible transgenic lines. When compared the viral resistance between SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants, soybean transgenic plants with the HC-Pro gene using RNAi strategy showed much stronger and higher frequency of viral resistance.

Bacillus thuringiensis(Bt) crystal protein (Cry1Ac) genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis(Bt) crystal protein genes. We transformed three difference Cry1Ac (Cry1Ac and two modified Cry1Ac) genes into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR, and RT-PCR. We generated 30 independent lines of transgenic soybean plants. Analysis of the flanking sequences isolated by Inverse PCR revealed complex T-DNA insertion patterns and preferential integration of T-DNA into the intergenic spacer region of the soybean genome. We found 5 different intergenic transgenic soybean lines of soybean genome. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis, physiology test, and agronomic characters are investigating.

Totally, 26 collections, 17 from Korea and 9 from China, were investigated for their sequences of 5S rDNA, especially the non-transcribed spacers (NTSs). Sequences of 5S rDNA were isolated by PCR using the primers, 5s-rRNA1 and 5s-rRNA2. Genomic DNA PCR produced single amplification of 300, 330, or 350 base pair fragments. Sequence analysis revealed that all inserts contained the part of 5S rDNA gene sequence and the full length of the NTS region. Three different sizes of the fragments were confirmed due to different size of NTS and their length were 300bp, 330bp and 350bp, respectively. Among 17 Korean foxtail millets tested, 14 collections showed single 300bp amplification. Longest fragment amplification, 350bp, was obtained only from the foxtail millet from China origin, even though 2 of them include 300bp fragment. CLUSTALW multiple alignments of 26 foxtail millets clearly revealed 4 areas with certain degree of sequence heterogeneity (region I, II, III, IV). Among 4 boxed areas, foxtail millet genotypes from China have distinct insertion especially in region III. Five of them have extra insertion of sequence and their additional sequences were either 45 or 48 base pair. Three Korean foxtail millets have 32 bp insertion. Other 8 Korean collections have short insert sequences (6 to 8 bp), 3 with 8 bp and 5 with 6 bp. In addition to insert, deletion sequences were also confirmed as major deletion was observed in region II of Chinese collection. The size of deletion was 7 bp long. According to phylogenic tree constructed using MEGA4 program, clear grouping was not revealed. To obtain more convincing results various collections from many countries should be obtained and analyzed to distinguish different germplasm from different origin.

Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.

Cry1Ac protein is known as one of toxin crystal proteins synthesized from Bacillus thuringenesis that plays a critical role for the insect resistance. Recently, cry1Ac genes have introduced into many plants in general and soybean as well. However, the gene expression of cry1Ac genes in transgenic plants remains low that need to be improved. Several mutations we reintroduced into the cry1Ac genes in order to enhance the insecticidal effect. In this study, the cry1Ac with mutant #2, #11 and #16 were transformed into Kwangan, a Korean soybean variety by using the “half-seed” method. The plant lets carrying modified cry1Ac genes were primarily selected on media containing Phosphinothricine (PPT), a bar selective agent and Basta leaf painting. Then, the presence of introduced genes in T0 plants and the gene expression were investigated by PCR, RT-PCR and Real-time PCR. PCR and RT-PCR analysis showed expression of bar and cry1Ac genes from tested transgenic soybean plants. The number of copy of bar gene ranged from 1 to 3 by Real-time PCR analysis. These results provided a fundamental back ground for our further experiments: Confirmation of the gene expression by Southern blot and identification of the function of modified cry1Ac by insect bioessays.

Bacillus thuringiensis (Bt) crystal protein genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis (Bt) crystal protein genes. These genes were transformed into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR and RT-PCR. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis and physiology test and agronomic characters are investigating.

Korean soybean variety Kwangan was transformed with ORE7 gene using highly efficient soybean transformation system. The gene is known to exhibit a delayed leaf senescence phenotype in Arabidopsis. To confirm phenotypic characterization of leaf senescence for non-transgenic (NT) and transgenic plants, we transplanted T1 transgenic lines 7, 9, 14 and 15 together with two negative controls (NT and EV) in greenhouse. As a result, line 15 showed dramatic phenotypic characterization of yield increase and senescence delay. In addition, to investigate the agriculture traits for transgenic plants with leaf senescence delaying, T2 transgenic lines and two negative controls were transplanted on GMO fields in Ochang and harvested T3 seeds (2010). Most transgenic lines showed higher total seed weigh than NT. Especially, total seed weight of line 15 was increased by about 180% and 120% compared with the NT and EV, respectively. Therefore, we carried out the second field experiments with T3 transgenic line 15 and NT in Ochang (2011). A total of 117 transgenic plants were divided into two groups, senescence delaying (64 out of 117 plants) and increased yield (53 out of 117 plants), by transcript level of ORE7 gene. Interestingly, among increased yield plants, total seed weight of each 7 plants were increased by more than 200% compared with NT.