cerevisiae and P. methanolica in this study, or crops may bring about enhanced growth and production of useful products under adverse culture conditions. Overexpressing enzymes involved in redox reaction in crops, such as superoxide dismutase  Proteasomal inhibitor and
glutathione peroxidase  has resulted in enhanced tolerance to salt and other stress. Methods Yeast strains and growth conditions The yeast strains used in this work included D. hansenii strain BCRC No. 21947, isolated from Hsilo County, Taiwan, S. cerevisiae Neo Type strain Y1 BCRC No. 21447 from brewer’s top yeast, obtained from FIRDI (Food Industry Research and Development Institute, Hsin-chu City, Taiwan), and P. methanolica strain PMAD11 genotype ade2-11, obtained from Invitrogen, U.S.A. D. hansenii was cultured at 24°C in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% dextrose) while S. cerevisiae and P. methanolica were cultured at 28°C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) and YPAD medium (1% yeast extract, 2% peptone, 2% dextrose, 0.01% adenine), respectively. RNA extraction and poly(A+) RNA purification click here Total RNA was extracted with a modified hot phenol protocol . Poly (A+) RNA was isolated from total RNA using Mag-Net mRNA Isolation Kit according to the manufacturer’s instruction (Amresco, Inc. USA). Concentration of RNA was determined using a
NanoDrop spectrophotometer (NanoDrop, Wilmington, USA). RNA quality was verified by electrophoresis on 1.5% formaldehyde agarose gel and stained with ethidium bromide. www.selleckchem.com/products/MK-1775.html subtractive hybridization and construction of subtracted cDNA library Subtractive hybridization was performed using PCR-select cDNA Subtraction Kit (Clontech, Palo Alto, CA, U.S.A.). For screening of differentially upregulated genes, cDNA synthesized from the 2.5 M NaCl treated yeast cells for
24 min was used as the tester while that from non-treated cells served as the driver. The PCR products of forward new subtraction were subcloned into the pGEMR-T Easy Vector (Promega, USA). Competent cells of E. coli (XL-Blue) was transfected with the plasmids and grown on LB-agar medium containing 5-bromo-4-chloro-3-indolyl-b-d-galactoside (X-gal) (Sigma, U.S.A.), isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma, U.S.A.) and ampicillin. Individual white colonies with insert DNA were randomly picked for further analysis. Sequencing and sequence analysis White clones from the forward subtractive hybridization libraries were sequenced with the universal T7 or SP6 sequencing primers using an automatic DNA sequencer (3100 Genetic Analyzer, ABI, U.S.A). All inserted sequences were queried for similarity through the NCBI database using BLASTX sequence comparison software http://www.ncbi.nlm.nih.gov/BLAST. Quantification of DhAHP by quantitative real-time PCR (Q-RT-PCR) Total RNA isolated from yeast cells treated with NaCl for various time intervals was first treated with DNase I (Promega, U.S.A.