Computational protocol: The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity

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Protocol publication

[…] DNA and RNA analysis used standard techniques . Fungal DNA or RNA was prepared by first grinding lyophilized mycelium into a fine powder with a mortar and pestle and then dispersing it in extraction buffer as described by Cenis . DNA for Southern hybridization experiments was prepared following the protocol of Doyle and Doyle . For Southern blot analysis, genomic DNA was digested with the indicated restriction enzymes (Fermentas GmbH, St. Leon-Rot, Germany), fractionated in 1% (w/v) agarose gels, and transferred to Nytran® nylon transfer membranes (Whatman Inc., Sanford, ME, USA) by downward blotting . 32P-labelled probes were prepared using the random oligomer-primer method and membranes were hybridized according to the protocol of Sambrook et al. .Total F. fujikuroi RNA was isolated using the RNAgents total RNA isolation kit (Promega GmbH, Mannheim, Germany). Samples of 20 µg of total RNA were transferred to Hybond-N+ membranes after electrophoresis on a 1% (w/v) agarose gel containing 1% (v/v) formaldehyde, according to Sambrook et al. . Northern blot hybridizations were accomplished by the method of Church and Gilbert . cDNA was synthesized from 1 µg of total RNA and the SuperScript II reverse transcriptase (Invitrogen, Groningen, The Netherlands) according to the manufacturer's instructions.All primers used for PCR were obtained from Eurofins GmbH (Ebersberg, Germany) (). PCR reactions contained 25 ng DNA, 5 pmol of each primer, 200 nm dNTPs, and 1 unit of BioTherm™DNA polymerase (GeneCraft GmbH, Lüdinghausen, Germany) and were initiated with a 4 min soak at 94°C followed by 36 cycles of 1 min at 94°C, 1 min at 56 to 65°C, 1–3 min at 70°C, and a final soak for 10 min at 70°C. PCR products were cloned into pCR®2.1-TOPO® vector using the TOPO TA Cloning® kit (Invitrogen, Groningen, The Netherlands) and transformed into Escherichia coli (Invitrogen). Plasmid DNA from E. coli was extracted using the GeneJET™ Plasmid Miniprep Kit (Fermentas GmbH, St. Leon-Rot, Germany) and sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit and the ABI Prism® 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according the manufacturer's instructions. DNA and protein sequence alignments were done with DNA STAR (Madison, WI, USA). Sequence homology searches were performed using the NCBI database server. Protein homology was based on BlastX searches . Phylogenetic analysis was performed using the web-based tool at . The nucleotide and protein sequences were deposited in GenBank under accession number HE614113 (ppt1), HE614114 (aar1), HE614115 (fet1), HE614116 (ftr1), HE614117 (ftr2), HE614118 (fet2), HE614119 (ftr3), HE614120 (fet3), HE614121 (nps2), HE614122 (nps6) and HE614123 (sre1), respectively. […]

Pipeline specifications

Software tools BLASTX,
Application Phylogenetics
Organisms Oryza sativa, Aspergillus fumigatus
Chemicals Iron