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ABSTRACT: BACKGROUND: Nitrogen limitation can induce neutral lipid accumulation in microalgae, as well as inhibiting their growth. Therefore, to obtain cultures with both high biomass and high lipid contents, and explore the lipid accumulation mechanisms, we implemented nitrogen deprivation in a model diatom Phaeodactylum tricornutum at late exponential phase. RESULTS: Neutral lipid contents per cell subsequently increased 2.4-fold, both the number and total volume of oil bodies increased markedly, and cell density rose slightly. Transcriptional profile analyzed by RNA-Seq showed that expression levels of 1213 genes (including key carbon fixation, TCA cycle, glycerolipid metabolism and nitrogen assimilation genes) increased, with a false discovery rate cut-off of 0.001, under N deprivation. However, most light harvesting complex genes were down-regulated, extensive degradation of chloroplast membranes was observed under an electron microscope, and photosynthetic efficiency declined. Further identification of lipid classes showed that levels of MGDG and DGDG, the main lipid components of chloroplast membranes, dramatically decreased and triacylglycerol (TAG) levels significantly rose, indicating that intracellular membrane remodeling substantially contributed to the neutral lipid accumulation. CONCLUSIONS: Our findings shed light on the molecular mechanisms of neutral lipid accumulation and the key genes involved in lipid metabolism in diatoms. They also provide indications of possible strategies for improving microalgal biodiesel production.
Biotechnology for Biofuels 05/2013; 6(1):67. · 6.09 Impact Factor
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ABSTRACT: The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.
Biotechnology and Applied Biochemistry 04/2013; · 1.53 Impact Factor
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ABSTRACT: Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.
BioTechniques 06/2012; · 2.67 Impact Factor
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ABSTRACT: The effect of rhizobacterium Burkholderia sp. strain R456 on the inhibition of Rhizoctonia solani, sheath blight of rice was examined. Results from this study indicated that strain R456 not only suppressed the in vitro
mycelial growth of R. solani, but also reduced the incidence and severity of rice sheath blight under greenhouse conditions. However, similar to plant
pathogenic strain LMG 1222T of Burkholderia cepacia, the type species of the genus, infiltration of tobacco leaves with cell suspension of strain R456 resulted in typical hypersensitivity
reactions while the two bacterial strains were unable to cause disease symptoms on rice seedlings. The fatty acid methyl ester
profile, sole carbon source utilization, and biochemical tests confirmed that the antagonistic rhizobacterium R456 is a member
of the genus Burkholderia. Furthermore, strain R456 was differentiated from B. cepacia LMG 1222T and was identified as Burkholderia seminalis based on recA gene sequence analysis and multilocus sequence typing. In addition, this rhizobacterium had a lower proteolytic activity
compared with that of the pathogenic B. cepacia LMG 1222T while no cblA and esmR marker genes were detected for the two bacterial strains. Overall, this is the first characterization of rhizobacterium B. seminalis that protected rice seedlings from infection by R. solani.
KeywordsAntagonism–
Burkholderia seminalis
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Rhizoctonia solani
–Rice sheath blight–Characterization
World Journal of Microbiology and Biotechnology 04/2012; 27(10):2305-2313. · 1.53 Impact Factor
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ABSTRACT: The in vitro antibacterial activity and mechanism of action of two kinds of acid-soluble chitosan and one water-soluble chitosan against apricot fruit rot pathogen Burkholderia seminalis was examined in this study. Results showed that water-soluble chitosan displayed limited antibacterial activity at four tested concentrations. However, two kinds of acid-soluble chitosan solution at 2.0 mg/mL had strong antibacterial activity against B. seminalis although weak antibacterial activity was observed at a concentration lower than 1 mg/mL. The antibacterial activity of acid-soluble chitosan may be due to membrane disruption, cell lysis, abnormal osmotic pressure, and additional chitosan coating around the bacteria based on integrity of cell membranes test, out membrane permeability assays and transmission electron microscopy observation. In addition, biofilm biomass were markedly reduced after treating with two kinds of acid-soluble chitosan at concentrations of 2.0 and 1.0 mg/mL for 3 and 12 h, indicating the importance of biofilm formation in the antibacterial mechanism of chitosan. Overall, the results clearly indicated that two kinds of acid-soluble chitosan had a potential to control the contamination of apricot fruits caused by B. seminalis.
Carbohydrate research 08/2011; 346(11):1294-301. · 2.03 Impact Factor
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ABSTRACT: To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry.
A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software.
Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL.
pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.
Biomedical and Environmental Sciences 10/2009; 22(5):419-22. · 1.35 Impact Factor
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ABSTRACT: The wood sawdust from Chinese fir (Cunninghamia lanceolata) exhibited stronger inhibition on the growth of Alexandrium tamarense than those from alder (Alnus cremastogyne), pine (Pinus massoniana), birch (Betula alnoides) and sapele (Entandrophragma cylindricum). The water extract, acetone-water extract and essential oil from fir sawdust were all shown to inhibit the growth of A. tamarense. The inhibition of fir essential oil was the strongest among all the above wood sources while the half effective concentration was only 0.65 mg/L. These results suggested that the fir essential oil may play an important role in the algicidal effect of Chinese fir.
Bulletin of Environmental Contamination and Toxicology 08/2009; 83(4):537-41. · 1.02 Impact Factor
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ABSTRACT: A laboratory experiment with orthogonal design was conducted to study the effects of factors salinity, temperature, and light intensity on the growth and toxin production of Chattonella marina. Three levels of salinity (22, 33, and 45), temperature (20 degrees C, 25 degrees C, and 30 degrees C) and light intensity (2000, 3000, and 4500 lx) were installed. In all treatments, the three factors had no significant effects on the growth of C. marina, but salinity significantly affected the toxin production of C. marina. Under salinity 45, temperature 30 degrees C and light intensity 2000 lx, C. marina had the maximal growth rate; under salinity 22, temperature 20 degrees C and light intensity 4500 lx, the toxin production of C. marina was the maximal. Low salinity was not favorable to the C. marina growth but favorable to its haemolytic toxin production. When the growth of C. marina was limited, its haemolytic toxin production increased.
Ying yong sheng tai xue bao = The journal of applied ecology / Zhongguo sheng tai xue xue hui, Zhongguo ke xue yuan Shenyang ying yong sheng tai yan jiu suo zhu ban 05/2009; 20(5):1190-5.
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ABSTRACT: Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.
Journal of Zhejiang University SCIENCE B 11/2008; 9(10):823-8. · 1.10 Impact Factor
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ABSTRACT: This study was conducted to determine the efficacy of chitosan at different concentrations with various intrinsic viscosities alone, and in its combination with a yeast antagonist Cryptococcus laurentii in reducing the blue mold rot caused by Penicillium expansum in apple fruit. The results indicated that application of chitosan alone was effective in inhibiting the blue mold rot in apple fruit wounds, especially with the high concentrations and low viscosities. But its efficacy was declining with the incubation time so that chitosan alone could not provide enduring protection of apple fruit from P. expansum infections. When applied at the concentration range from 0.001 to 0.1% (wt/vol), chitosan did not influence the population growth of C. laurentii in vivo, whereas it markedly repressed the yeast growth as its concentrations were increased up to 0.25% (wt/vol) or higher. Moreover, combination of chitosan and C. laurentii resulted in a synergistic inhibition of the blue mold rot, being the most effective at the optimal concentration of 0.1% of chitosan with the lowest viscosity (12 cP). The possible mode of action of the combination of chitosan and C. laurentii was discussed.
International Journal of Food Microbiology 04/2007; 114(3):261-6. · 3.33 Impact Factor
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ABSTRACT: Effects of toxic Alexandrium species on Prorocentrum donghaiense were studied in co-cultures and filtrates in order to shed some light on the competition between the co-occurring harmful algae. The three live cultures of Alexandrium tamarense and Alexandrium minutum negatively affected the growth of P. donghaiense, while only A. minutum was affected by P. donghaiense when they were in 1:3 ratio of cells. Each species was affected by the co-occurring algae as could be observed by microscopy. The allelopathic effects observed in co-culture did not correlate with the measured content of the intracellular PSP toxins but matched the observed hemolytic properties of the culture filtrates, suggesting that certain hemolytic substances other than PSP toxins are responsible for the allelopathic effect of Alexandrium.
Harmful Algae 10(1):116-120. · 3.08 Impact Factor
