Dong-Woog Choi

Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites

Methyl jasmonate (MeJA) treatment increases the levels of plant secondary metabolites, including ginsenosides, which are considered to be the main active compounds in ginseng (Panax ginseng C.A. Meyer). To create a ginseng gene resource that contains the genes involved in the biosynthesis of secondary metabolites, including ginsenosides, we generated 3,134 expression sequence tags (ESTs) from MeJA-treated ginseng hairy roots. These ESTs assembled into 370 clusters and 1,680 singletons. Genes yielding highly abundant transcripts were those encoding proteins involved in fatty acid desaturation, the defense response, and the biosynthesis of secondary metabolites. Analysis of the latter group revealed a number of genes that may be involved in the biosynthesis of ginsenosides, namely, oxidosqualene cyclase (OSC), cytochrome P450, and glycosyltransferase. A novel OSC gene was also identified by this analysis. RNA gel blot analysis confirmed that transcription of this OSC gene, along with squalene synthase (SS) and squalene epoxidase (SE) gene transcription, is increased by MeJA treatment. This ginseng EST data set will also provide important information on the genes that are involved in the biosynthesis of other secondary metabolites and the genes that are responsive to MeJA treatment.

High temperature thermoelectric properties of p- and n-type β-FeSi2 with some dopants

Abstract Cr doped p-type and Co doped n-type β-FeSi2 alloys have been prepared using a powder metallurgy technique. Then the effects of Cu and Ge additions on their thermoelectric properties were investigated. Thermoelectric power α, electrical conductivity, σ, and thermal conductivity, κ were measured in a temperature range from 373 to 973 K. Their thermoelectric power is found to decrease with increasing an amount of both Cu and Ge in both type of β-FeSi2. In contrast, their electrical conductivity exhibit opposite tendency with respect to the amount of both dopants. Their thermal conductivity is more effectively reduced in the Ge addition to them than that in the Cu addition. The figure of merit, Z of binary β-FeSi2 is 0.19×10−4/K, and increases by the addition of Cr, Co, and Ge. The maximum figure of merit, Z of 1.3×10−4/K was obtained in Fe0.95Co0.05Si1.958Ge0.042 at 845 K.

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts

Summary The SNAT gene of the red alga Pyropia yezoensis is chloroplast encoded, suggesting that plant SNAT genes evolved directly from cyanobacteria to red algae via endosymbiosis and thereafter were transferred to the nucleus.

Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas.

Heat shock proteins and molecular chaperones are key components contributing to survival in the abiotic stress response. Porphyra seriata grows on intertidal rocks exposed to dynamic environmental changes associated with the turning tides, including desiccation and heat stress. Analysis of the ESTs of P. seriata allows us to identify the nine HSP cDNAs, which are predicted to be PsHSP90, three PsHSP70, PsHSP40 and PsHSP20, and three 5′-truncated HSP cDNAs. RT–PCR results show that most of the PsHSP transcripts were detected under normal cell growth conditions as well as heat stress, with the exception of two cDNAs. In particular, PsHSP70b and PsHSP20 transcripts were upregulated by heat stress. When the putative mitochondrial PsHSP70b was introduced and overexpressed in Chlamydomonas, transformed Chlamydomonas evidenced higher rates of survival and growth than those of the wild type under heat stress conditions. Constitutive overexpression of the PsHSP70b gene increases the transcription of the HSF1 as well as the CrHSP20 and CrHSP70 gene. These results indicate that PsHSP70b is involved in tolerance to heat stress and the effects on transcription of the CrHSP20 and CrHSP70 genes.

IDENTIFICATION OF THE HIGH-TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonasreinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropiatenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropiatenera/Chlamydomonasreinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.

Sequence variability and expression characteristics of the ginseng (Panax ginseng C.A. Meyer) dehydrin gene family

The dehydrins (DHN) are a family of late embryo abundant (LEA D-11) proteins, which accumulate during the late stage of seed development or under low temperature or water deficient conditions. They are believed to play a protective role in freezing and drought tolerance. The dehydrin genes exist as multi-gene families. Here, we have identified 9 unique dehydrin genes from Korean ginseng (Panax ginseng C.A. Meyer), a typical medicinal plant Among these,PgDhn1 andPgDhn2 encode for YSK3- and KS-type dehydrins, respectively, and are very abundant. Gene expression analyses revealed that the majority of thePgDhn gene transcripts are detected under cold, as well as dehydration conditions. The exceptions arePgDhn5 and PgDhn9—the former being unresponsive to cold treatment, and the latter exhibiting only seed-specific expression. We also identified an alternative transcript of thePgDhn2 gene that harbors an intron in its 3’-untranslated region. Our results may prove useful in further studies ofDhn genes, including investigations into the mechanisms underlying gene expression, the nature of their variations, and their physiological functions.

GUS Gene expression and plant regeneration via somatic embryogenesis in cucumber (Cucumis sativus L.)

One of the limitation for Agrobacterium-mediated transformation via organogenesis from cotyledon explants routinely in cucumber is the production of chimeric plants. To overcome the limitation, Agrobacterium-mediated transformation system via somatic embryogenesis from hypocotyl explants of cucumber (c.v., Eunsung) on the selection medium with paromomycin as antibiotics was developed. The hypocotyl explants were inoculated with Agrobacterium tumefaciens strain EHA101 carrying binary vector pPTN290; then were subsequently cultured on the following media: co-cultivation medium for 2 days, selection medium for days, and regeneration medium. The T-DNA of the vector (pPTN290) carried two cassettes, Ubi promoter-gus gene as reporter and 35S promoter-nptll gene conferring resistance to paromomycin as selectable agent. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to paromomycin indicated by the growth of putative transgenic calli on selection medium amended with 100mg/L paromomycin, and GUS gene expression. Forty eight clones (5.2%) with GUS gene expressed of 56 callus clones with resistance to paromomycin were independently obtained from 928 explants inoculated. Of 48 clones, transgenic plants were only regenerated from 5 clones (0.5%) at low frequency. The histochemical GUS assay in the transgenic seeds () also revealed that the gus gene was successfully integrated and segregated into each genome of transgenic cucumber.

Screening of Soybean Recombinant Inbred Lines for High Competence Somatic Embryogenesis

Cotyledonary explants from immature zygotic embryos of each 85 recombinant inbred lines (RILs) were cultured on medium containing MS salts, B5 vitamins, 40 mg 2,4-dichlorophenoxyacetic acid and 30 g sucrose. Frequency of somatic embryo formation on cotyledonary explants showed in thirty-six lines(＜10%), in thirty-seven lines (11~49%), in nine lines (50~89%), and in three lines(>90%), respectively, The highest frequency (up to 90%) and number (6.36 per cotyledon) of somatic embryos were obtained from lines of KM1010, KM1032 and KM1064. Primary somatic embryos produced from three lines produced numerous secondary somatic embryos on the surfaces, which were subcultured for over one year. Upon transfer to maturation and conversion medium (Komatsuda, 1992), somatic embryos converted to plantlets at a frequency of approximately 25%.

Production of Human Serum Albumin in Chloroplast-Transformed Tobacco Plants

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5`UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.