教師研究

教師研究

  • 徐泰浩

    篇  名

    Potential antidiabetic activity of extracellular polysaccharides in submerged fermentation culture of Coriolus versicolor LH1

    摘  要

    The separation and purification of extracellular polysaccharides from Coriolus versicolor LH1 were investigated along with their _-glucosidase inhibition properties. Three polysaccharide fractions (ePS-F2-1,ePS-F3-1, and ePS-F4-1) were separated from the culture medium of LH1 using a DEAE anion-exchange column and a SephadexTM G-50 gel filtration column. Their chemical compositions was determined. On the basis of an _-glucosidase inhibition assay, the enzyme inhibition activities of ePS-F2-1, ePS-F3-1,and ePS-F4-1 were investigated. Among these, ePS-F4-1 had the highest enzyme inhibition effects on_-glucosidase. According to the results of the chemical component analysis, ePS-F3-1 and ePS-F4-1 are the polysaccharides which are combined with triterpenoides, and ePS-F2-1 is complexed with proteins and triterpenoides.

    全文連結

    徐泰浩.pdf

  • 何偉真

    篇  名

    Cucurbitacin E Induces G2/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells

    摘  要

    Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨm) and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G2/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 1 (CDK1) and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID) and a loss of ΔΨm, resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1) and apoptosis-inducing factor (AIF), and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G2/M phase arrest and apoptosis of T24 cells.

    全文連結

    何偉真.pdf

  • 柯文慶

    篇  名

    Optimized Extraction Method of Acetic Acid in Vinegar and Its Effect on SNIF-NMR Analysis to Control the Authenticity of Vinegar

    摘  要

    Site-specific natural isotope fractionation by nuclear magnetic resonance (SNIF-NMR) is an accurate method for examining food adulteration. SNIF-NMR can be used to determine the ratio of deuterium to hydrogen (D/H) of a specific molecule position. Because the quantity of D differs between synthetic and fermented acetic acids, the method can be used to accurately monitor food adulteration. However,the effect of pretreated food on the results of SNIFNMR has rarely been discussed. We present an extractive distillation method to increase the purity of acetic acid for fractionation; we used an orthogonal array experimental design to determine the optimal extraction conditions. We discuss the influence of extract solvents and sample concentration on the (D/H)CH3 values calculated from the NMR results. The optimal conditions for extracting acetic acid were found to be a sample-to-solvent ratio of 1:1, seven extractions, and an extraction time of 15 min. The extraction rates for acetic acid were 93.65% and 80.57% when ethyl acetate and diethyl ether, respectively, were used. The acetic acid concentration of the ethyl acetate- and diethyl etherextracted samples was further improved by distillation from 5 g/100 mL to 33.84 and 51.65 g/100 mL, respectively.

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    柯文慶.pdf

  • 涂瑞澤

    篇  名

    A green peptide synthesis?Using a magnetic biocatalyst in a stirred-tank bioreactor

    摘  要

    The optimizationofsynthesisdipeptidederivative, N-Ac-Phe-Gly-NH2, catalyzedbyimmobilized a-chymotrypsin(a-chymotrypsinimmobilizedonFe3O4-chitosan; a-chymotrypsin-Fe3O4-CS)ina stirred-tankbioreactorisevaluatedbytheexperimentaldesign.Threestate-of-the-artgreentechnol- ogies (i.e.,enzymecatalysis,immobilizedenzyme,andmagneticbioreactor)arecombinedinthisstudy. Responsesurfacemethodology(RSM)witha3-factor-3-levelBox-Behnkendesignisemployed to evaluatetheeffectsofthesynthesisparameters,includingreactiontime(30–90min),temperature (25–45 1C) andpH(7–9).Amodelforthesynthesisisdevelopedandtheoptimumconditionsare predictedtobeareactiontimeof92.3min,areactiontemperatureof36.2 1C andpH8.7.An experimentisperformedundertheseoptimumconditionsandayieldof82.26%isobtained.After the reaction,magneticallyimmobilized a-chymotrypsinisrecoveredbyamagneticfieldandused repeatedlytosynthesizedipeptideattheoptimumconditions.Ayieldofaround82%ofdipeptideis obtainedinthefirst3repeatedbatchesandtheyieldover70%isretainedaftertenbatches.Itis expectedthatthisapproachhasgreatpotentialinindustryforthelarge-scaleproductionofthe dipeptidederivative.

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    涂瑞澤.pdf

  • 涂耀國

    篇  名

    Effects of pH on Electrocatalytic Activity of Carbon Nanotubes in Poly(acrylic acid) Composites Used for Electrochemical Selective Detection of Uric Acid and Dopamine in the Presence of Ascorbic Acid

    摘  要

    Effects of pH on dispersion of carbon nanotubes (CNT) in poly(acrylic acid) (PAA) composites and on electrocatalytic activity of CNT toward oxidation reactions of uric acid (UA) at the composite-modified glassy carbon electrode (GCE) were investigated. Cyclic voltammetry data found that CNT in PAA could electrocatalyze the oxidation reactions of UA as indicated by a significant increase in the anodic peak current (Ipa). Better dispersion of CNT in the PAA cast film exhibited a higher Ipa and electrocatalytic activity. The Ipa was increased with increasing CNT content, with the highest increasing rate from pH 7 at which the electrode was modified by the composite. For a given pH, CNT had higher electrocatalytic activity than fCNT (the functionalized CNT). In the presence of an excess of ascorbic acid, UA and dopamine (DA) could be simultaneously detected by the PAA/CNT 10/2.5 of pH 7-modified GCE. The sensitivity for simultaneous detection of DA was higher than that for UA. The linear range for the DA detection was 0.04−6 μM and for the UA detection was 0.04−3 μM.

    全文連結

    涂耀國.pdf

  • 吳淑姿

    篇  名

    Continuous production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Effects of C/N ratio and dilution rate on HB/HV ratio

    摘  要

    Abstract−Ralstonia eutropha was cultivated in a continuous stirred fermenter with various C/N ratios (20, 30, and40), dilution rates, and organic salt substrates (sodium propionate or sodium valerate) to explore the microbial growth and the poly(3HB-co-3HV) accumulation. When sodium propionate was used as the secondary carbon source, the HB/HV molar ratio at various C/N ratios and dilution rates did not change appreciably (approximately 90 : 10). The highest poly(3HB-co-3HV) content in biomass (41.8%) and poly(3HB-co-3HV) productivity (0.100 g/(L•h)) occurred under the condition with a C/N ratio of 20 and dilution rate of 0.06 h−1. When sodium valerate was used as the secondary carbon source, the productivity of poly(3HB-co-3HV) increased with increasing dilution rate for the C/N ratio of 30 and 40. The average HB/HV molar ratio ranged from 48 : 52 to 78 : 32. The feeding of sodium valerate promoted the accumulation of HV better than feeding sodium propionate did. This study shows that a potential strategy of manipulating by both C/N ratio and dilution rate could be used to control the HV unit fraction in poly(3HB-co-3HV) in a continuous cultivation.

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    吳淑姿.pdf

  • 簡宏堅

    篇  名

    Production of N-acetyl-d-neuraminic acid by recombinant whole cells expressing Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase and Escherichia coli N-acetyl-d-neuraminic acid lyase

    摘  要

    N-acetyl-d-neuraminic acid (NeuAc; sialic acid) is a precursor for the manufacture of many pharmaceutical drugs, such as anti-influenza virus agents. To develop a whole cell process for NeuAc production, genes of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase (bage) and Escherichia coli N-acetyl-d-neuraminic acid lyase (nanA) were cloned and expressed in E. coli BL21 (DE3). The expressed bGlcNAc 2-epimerase was purified from the crude cell extract of IPTG-induced E. coli BL21 (DE3) (pET-bage) to homogeneity by nickel-chelate chromatography.The molecular mass of the purified bGlcNAc 2-epimerase was determined to be 42 kDa by SDS-PAGE. The pH and temperature optima of the recombinant bGlcNAc 2-epimerase were pH 7.0 and 50 ◦C, respectively, and only needs 20 _m ATP for maximal activity. The specific activity of bGlcNAc 2-epimerase (124Umg−1 protein) for the conversion of N-acetyl-d-glucosamine to N-acetyl-d-manosamine was about four-fold higher than that of porcine N-acetyl-d-glucosamine 2-epimerase. A stirred glass vessel containing transformed E. coli cells expressing age gene from Anabaena sp. CH1 and NeuAc lyase gene from E. coli NovaBlue separately was used for the conversion of Glc-NAc and pyruvate to NeuAc. A maximal productivity of 10.2 g NeuAc l−1 h−1 with 33.3% conversion yield from GlcNAc could be obtained in a 12-h reaction. The recombinant E. coli cells can be reused for more than eight cycles with a productivity of >8.0 g NeuAc L−1 h−1. In this process, the expensive activator, ATP, necessary for maximal activity of GlcNAc 2-epimerase in free enzyme system can be omitted.

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    簡宏堅.pdf

  • 張世良

    篇  名

    Successful treatment of methemoglobinemia in an elderly couple with severe cyanosis: two case reports

    摘  要

    Introduction
    Methemoglobinemia should be considered in all cyanotic patients who remain unresponsive to oxygen therapy. Rapid diagnosis is very important in emergency cases. Here, we present the cases of two patients, a married couple, admitted to our hospital with methemoglobinemia after exposure to sodium nitrite. Case presentation
    Two patients, a married couple, presented with methemoglobinemia. The 72-year-old Taiwanese man and 68-year-old Taiwanese woman were referred to our hospital with dizziness and tachypnea. On examination, their mucous membranes were cyanotic, and their blood samples showed the classic ‘chocolate brown’ appearance. The man also reported having experienced twitching of his right arm for a few minutes before arrival at the hospital. The symptoms of both patients failed to improve in response to supplemental oxygen delivered via oxygen masks, although the arterial blood gas data of these patients were normal and their pulse oximetry showed oxyhemoglobin levels of approximately 85%. A carbon monoxide-oximeter showed that the man’s methemoglobin concentration was 48.3%,and the woman’s was 36.4%. Methylene blue (100mg) was administered intravenously to both patients, and their symptoms improved dramatically. They were admitted to the intensive care unit and discharged three days later, without neurological sequelae.
    Conclusion
    Severe methemoglobinemia is a life-threatening condition and, if untreated, may result in death. Early diagnosis and appropriate antidotal treatment are crucial in treating this emergency situation.

    全文連結

    張世良.pdf

  • 洪淑嫻

    篇  名

    Forced flowering of pineapple (Ananas comosus cv. Tainon 17) in response to cold stress, ethephon and calcium carbide with or without activated charcoal

    摘  要

    Ethylene, a gaseous plant hormone, is responsible for the initiation of reproductive development in pineapple. Reproductive development can be forced in pineapple (Ananas comosus var. comosus) throughout the year with ethylene. Inhibition of natural flowering initiation with aviglycine [(S)-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride], an inhibitor of ethylene biosynthesis, provides evidence that reproductive development in response to cold stress and short daylength is also in response to ethylene production. We studied the effect of cold treatment of pineapple on ethylene production and flower induction by applying a short-term cold stress to stem apices. Shoot apices of pineapple treated with ice crystals also produced twice as much ethylene as.
    did those of control plants and significantly more than was produced by ‘‘D’’ leaf basal tissue. Moreover, pineapple plants treated four times with ice crystals or ice water were induced to flower under field conditions and the forcing efficiency, as evaluated by the percentages of inflorescence emergence and fruit harvest, was comparable to forcing with calcium carbide (CaC2) and ethephon. In another field experiment two applications of a 1.0% solution of CaC2 or 0.15% ethephon applied at 48 h intervals was sufficient to force reproductive development of ‘Tainon 17’. Furthermore,0.5 or 1.0% solutions of CaC2 supplemented with 0.5% activated charcoal (AC) significantly improved the forcing effectiveness of CaC2. This could/would make it possible to reduce the number or concentration, or both, of CaC2 required to effect forcing in pineapple.

    全文連結

    洪淑嫻.pdf

  • 吳建一

    篇  名

    Production of keratinolytic enzyme by an indigenous feather—degrading strain Bacillus cereus Wu2

    摘  要

    A novel feather-degrading microorganism was isolated from a poultry farm in Taiwan, and was identified Bacillus cereus Wu2 according to 16S rRNA sequencing. The isolated strain produces keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source. The experimental results indicated that the extra carbon sources (glucose, fructose, starch, sucrose, or lactose) could act as a catabolite repressor to the enzyme secretion or keratinolytic activity when keratinous substrates were employed as protein sources. However, addition of 2 g/L of NH4Cl to the feather medium increased the enzyme production. The optimum temperature and initial pH for enzyme production were 30_C and 7.0, respectively. The maximum yield of the enzyme was 1.75 kU/mL in the optimal chicken feather medium; this value was about 17-fold higher than the yield in the basal hair medium. The B. cereus Wu2 possessed disulfide reductase activity along with keratinolytic activity. The amino acid contents of feathers degradated by B. cereus Wu2 were higher, especially for lysine, methionine and threonine which were nutritionally essential amino acids and usually deficient in the feather meal. Thus, B. cereus Wu2 could be not only used to enhance the nutritional value of feather meal but is also a potential bioinoculant in agricultural environments.

    全文連結

    吳建一.pdf

  • 林芳儀

    篇  名

    Potential antidiabetic activity of extracellular polysaccharides in submerged fermentation culture of Coriolus versicolor LH1

    摘  要

    The separation and purification of extracellular polysaccharides from Coriolus versicolor LH1 were inves-tigated along with their _-glucosidase inhibition properties. Three polysaccharide fractions (ePS-F2-1, ePS-F3-1, and ePS-F4-1) were separated from the culture medium of LH1 using a DEAE anion-exchange column and a SephadexTM G-50 gel filtration column. Their chemical compositions was determined. On the basis of an _-glucosidase inhibition assay, the enzyme inhibition activities of ePS-F2-1, ePS-F3-1, and ePS-F4-1 were investigated. Among these, ePS-F4-1 had the highest enzyme inhibition effects on _-glucosidase. According to the results of the chemical component analysis, ePS-F3-1 and ePS-F4-1 are the polysaccharides which are combined with triterpenoides, and ePS-F2-1 is complexed with proteins and triterpenoides.

    全文連結

    林芳儀.pdf

  • 游志文

    篇  名

    Constitutive expression of a fungal glucose oxidase gene in transgenic tobacco confers chilling tolerance through the activation of antioxidative defence system

    摘  要

    cientific evidences in the literature have shown that plants treated exogenously with micromole concentration of hydrogen peroxide (H2O2) acquire abiotic stress tolerance potential, without substantial disturbances in the endogenous H2O2 pool. In this study, we enhanced the endogenous H2O2 content of tobacco (Nicotiana tabaccum L. cv. SR1) plants by the constitutive expression of a glucose oxidase (GO; EC 1.1.3.4) gene of Aspergillus niger and studied their cold tolerance level. Stable integration and expression of GO gene in the transgenic (T0–T2) tobacco lines were ascertained by molecular and biochemical tests. Production of functionally competent GO in transgenic plants was confirmed by the elevated levels of H2O2 in the transformed tissues. When three homozygous transgenic lines were exposed to different chilling temperatures for 12 h, the electrolyte conductivity was significantly lower in GO-expressing tobacco plants than the control plants; in particular, chilling protection was more prominent at -1_C. In addition, most transgenic lines recovered within a week when returned to normal culture conditions after -1_C–12 h cold stress. However, control plants displayed symptoms of chilling injuries such as necrosis of shoot tip, shoots and leaves, consequently plant death. The protective effect realized in the transgenic plants was comparable to cold-acclimatized wild tobacco. The chilling tolerance of transgenic lines was found associated, at least in part, with elevated levels of total antioxidant content, CAT and APX activities. Based on our findings, we predict that the transgenic expression of GO may be deployed to improve cold tolerance potential of higher plants.

    全文連結

    游志文.pdf

  • 張雲祥

    篇  名

    Feeding hermit crabs to shrimp broodstock increases their risk of WSSV infection

    摘  要

    White spot syndrome virus (WSSV) is a serious shrimp pathogen that has spread globally to all major shrimp farming areas, causing enormous economic losses. Here we investigate the role of hermit crabs in transmitting WSSV to Penaeus monodon brooders used in hatcheries in Vietnam. WSSV-free brooders became PCR-positive for WSSV within 2 to 14 d, and the source of infection was traced to hermit crabs being used as live feed. Challenging hermit crabs with WSSV confirmed their susceptibility to infection, but they remained tolerant to disease even at virus loads equivalent to those causing acute disease in shrimp. As PCR screening also suggests that WSSV infection occurs commonly in hermit crab populations in both Vietnam and Taiwan, their use as live feed for shrimp brooders is not recommended.

    全文連結

    張雲祥.pdf

  • 黃尉東

    篇  名

    Penaeus monodon TATA box-binding protein interacts with the white spot syndrome virus transactivator IE1 and promotes its transcriptional activity.

    摘  要

    We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused “squelching” of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1’s transactivation activity that can enhance viral gene expression and help in virus replication.

    全文連結

    黃尉東.pdf

  • 蔡孟峰

    篇  名

    ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors (CP Huang and MF Tsai contributed equally to this work.)

    摘  要

    Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1- positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.

    全文連結

    蔡孟峰.pdf

  • 劉淑瑛

    篇  名

    Wide spread of Tn2006 in an AbaR4-type resistance island among carbapenem- resistant Acinetobacter baumannii clinical isolates in Taiwan

    摘  要

    Carbapenem resistance in Acinetobacter baumannii is a global problem. The purpose of this study was to elucidate current resistance mechanisms of imipenem-resistant A. baumannii (IRAB) in Taiwan and their correlation with patient outcomes. Acinetobacter baumannii clinical isolates from two teaching hospitals in Taiwan were collected in 2009 and were examined by Etest for determination of the minimum inhibitory concentrations (MICs) of imipenem, ceftazidime and ceftriaxone. Primers specific for carbapenemase genes and upstream regions were designed for PCR amplification. Bacterial isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Clinical presentations of patients were analysed retrospectively. Upstream insertion sequence ISAba1 was found in 34 isolates that carried blaOXA-23, including 28 with transposon Tn2006 (ISAba1–blaOXA-23–ISAba1) in an AbaR4-type resistance island and 6 with Tn2008 (ISAba1–blaOXA-23), as well as in 8 isolates carrying ISAba1–blaOXA-51-like. All of these isolates expressed full resistance to imipenem (MIC > 32 mg/L). Forty-one different PFGE genotypes were found among 62 isolates. Tn2006 was found in 19 genotypes (46.3%), which is more common than ISAba1–blaOXA-51-like (12.2%) (P = 0.001). Prior use of carbapenems or extended-spectrum cephalosporins for ≥5 days was the only independent risk factor significantly associated with IRAB infection.
    (odds ratio = 361.175). Higher mortality was significantly associated with infection caused by IRAB and ISAba1–blaOXA-23-carrying strains compared with infection caused by imipenem-susceptible A. baumannii and ISAba1–blaOXA-51-like-carrying strains (P = 0.009 and 0.027, respectively). Tn2006 is currently the most common imipenem resistance determinant, which showed a higher ability to spread among A. baumannii and was associated with a higher mortality in IRAB-infected patients.

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  • 林重宏

    篇  名

    Cultivation of the Culinary-Medicinal Lung Oyster Mushroom, Pleurotus pulmonarius (Fr.) Quél. (Agaricomycetideae) on Grass Plants in Taiwan

    摘  要

    Cultivation of the culinary-medicinal Lung Oyster mushroom, Pleurotus pulmonarius, on the stalks of three grass plants, i.e., Panicum repens, Pennisetum purpureum, and Zea mays were investigated. The effects of various combinatorial substrates on mushroom mycelial growth and yield calculated as biological efficiency (BE) were determined. Among 9 experimental substrates, the most suitable substrate for mycelial growth was 45ZMS:45S, followed by 45PRS:45S; their mycelial growth rates were obviously quicker than that of the control substrate. The BEs of all the experimental substrates respectively containing P. repens stalk, P. purpureum stalk and Z. mays stalk were higher than that of the control (39.55%) during the 2.5 months of cultivation period. The best substrate in terms of BE was 60ZMS:30S (58.33%), followed by 45PRS:45S (57.16%), 45ZMS:45S (49.86%), and 30ZMS:60S (47.20%). Based on the BE of the tested substrates, Z. mays stalk appeared to be the best alternative material for the production of P. pulmonarius.

    全文連結

    林重宏.pdf

  • 余聰安

    篇  名

    Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct

    摘  要

    Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R0 plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R1 progeny of the two resistant R0 lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R1 plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

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    余聰安.pdf

  • 李泰林

    篇  名

    Construction of Candida albicans Tet-on tagging vectors with a Ura-Blaster cassette

    摘  要

    It has been difficult to develop molecular tools for studying the fungal pathogen Candida albicans because this species uses a non-standard genetic code and is diploid without a complete sexual cycle. Vector systems with regulatable promoters to produce conditional mutants, epitope tags for protein detection and recyclable selection markers are useful for functional study of genes. However, most currently available vectors contain only a subset of desired properties, which limits their application. To combine several useful properties in one vector, the vector pTET25 was initially modified into pTET25M, so that the URA3 gene flanked by dpl200 could be used repetitively. To enable more choices for cloning, a multiple cloning site was introduced at both ends of GFP in pTET25M. GFP expression was induced by doxycycline in a dose- and time-dependent manner when the plasmid was introduced into C. albicans with or without URA3. The applicability of the vectors was verified by constructing strains capable of expressing either the N-terminal GFP fusion of Cdc10 or the C-terminal GFP fusion of Cdc11. Additionally, by replacing the GFP gene of pTET25M with DNA sequence encoding Cdc10 with an epitope tag of six histidine residues at the C-terminus, doxycycline-induced expression of CDC10 was achieved when the expression vector was introduced into C. albicans. This new system allows for inducible expression of a desired C. albicans gene with the advantage of convenience of cloning. It also allows the presence of a recyclable URA3 marker and the detectable expression of fusion or epitope-tagged protein. Copyright 
    2010 John Wiley & Sons, Ltd.

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    李泰林.pdf

  • 江主惠

    篇  名

    Molecular Authentication of Dendrobium Species by Multiplex Polymerase Chain Reaction and Amplification Refractory Mutation System Analysis

    摘  要

    The genus Dendrobium is important in traditional Chinese herbal medicine, and the precise identification of Dendrobium species is critical for the treatment and for pharmacological research. In the present study, a ribosomal DNA (rDNA) internal transcribed spacer (ITS) region-based analysis was used to ascertain the phylogenetic relationship among 20 Dendrobium species. The lengths of the ITS regions among the 20 species ranged from 636 to 653 bp, and the identities of the rDNA regions among the different species ranged from 75.7% to 99.1%. The results also showed that the ITS1 and ITS2 regions exhibit more variation than the 5.8S rDNA. A phylogenetic tree derived from the ITS sequence indicated that six medicinal Dendrobium species, of which five are common medicinal plants in the Taiwan market, were closely related and shared a common clade. Multiplex polymerase chain reaction (PCR) amplification was successfully performed to identify the six medicinal Dendrobium species, and amplification refractory mutation system (ARMS) PCR was used to distinguish D. tosaense specifically from the 19 other Dendrobium species. The established PCR-based (multiplex and ARMS) analyses can be used for the authentication of the raw materials of medicinal Dendrobium from other species.

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    江主惠.pdf

  • 賴伯琦

    篇  名

    Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan

    摘  要

    Butachlor is the most commonly used herbicide.
    on paddy fields in Taiwan and throughout Southeast Asia.
    Since paddy fields provide habitat for pond breeding.
    amphibians, we examined growth, development, time to.
    metamorphosis, and survival of alpine cricket frog tadpoles.
    (Fejervarya limnocharis) exposed to environmentally.
    realistic concentrations of butachlor. We documented.
    negative impacts of butachlor on survival, development,.
    and time to metamorphosis, but not on tadpole growth. The.
    96 h LC50 for tadpoles was 0.87 mg/l, much lower than the.
    4.8 mg/l recommended dosage for application to paddy.
    fields. Even given the rapid breakdown of butachlor, tadpoles.
    would be exposed to concentrations in excess of their 96 h LC50 for an estimated 126 h. We also documented DNA damage (genotoxicity) in tadpoles exposed to butachlor at concentrations an order of magnitude less than the 4.8 mg/l recommended application rate. We did not find that butachlor depressed cholinesterase activity of tadpoles, unlike most organophosphorus insecticides. We conclude that butachlor is likely to have widespread negative impacts on amphibians occupying paddy fields with traditional herbicide application.

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    賴伯琦.pdf

  • 孫芳君

    篇  名

    Efficient production of an engineered apoptin from chicken anemia virus in a recombinant E.coli for tumor therapuetic applications

    摘  要

    Background
    Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV),has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.
    Results
    Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25°C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to ente apoptosis.
    Conclusions
    On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

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    孫芳君.pdf