Computational protocol: Mechanisms of action for 2-phenylethanol isolated from Kloeckera apiculata in control of Penicillium molds of citrus fruits

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Protocol publication

[…] Untreated (CK), and PEA-treated samples (PEA1 and PEA3 refer to 1.5 μL/mL-treated fungus for 1 hour and 3 hour, respectively), were harvested. Total RNA was extracted from P. italicum by Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA-Seq profiling was performed by Beijing Genomics Institute (Shenzhen, China). Brifly, mRNA was enriched by using oligo(dT) magnetic beads. The fragmentation buffer was added and mRNA was interrupted to approximately 200 bp. The first and second strand cDNA was synthesized by using reverse transcriptase and random hexamer-primer. Double-strand cDNA was purified with QiaQuick PCR extraction kit and washed with EB buffer for end repair and single nucleotide adenine addition. Finally, sequencing adapters were ligated to the fragments. The required fragments were purified by agarose gel electrophoresis and enriched by PCR amplification. The sequences of the library products were analyzed by using an Illumina HiSeq™ 2000.Clean reads were generated by removing adapter sequences, unknown bases more than 10% and low quality reads. Each tunnel generated 11.6 million reads with a sequencing length of 49 bp. All clean reads were then aligned to reference sequences of Penicillium chrysogenum Wisconsin 54–1255 by using SOAPaligner/soap2 []. Mismatches no more than 2 bases were allowed in the alignment. The expression level of gene (Additional file : Table S1) was calculated by using the RPKM (reads per kb per million reads) method []. Differentially expressed genes (DEG; Additional file : Table S1) in three samples were analyzed as described [–]. [...] Eight genes were chosen for confirmation by qRT-PCR with SYBR Premix Ex Taq™ (Takara, Japan). Primers for the chosen genes were designed with the Primer Express software (Applied Biosystems, USA) and are presented in Additional file . qRT-PCR for gene expression analysis was performed on a StepOne Real-time PCR System (Applied Biosystems, USA) using β-tubulin gene as an endogenous control. Briefly, the primers for the target gene and β-tubulin were diluted in the SYBER Mix (Applied Biosystems) and 20 μL of the reaction mix were added to each well. The reactions were performed with an initial incubation at 50°C for 2 min and at 95°C for 1 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The levels of gene expression were analyzed with StepOne Software v2.0. Zero template controls were included for each primer pair. Each PCR reaction was carried out in triplicate, and the data are presented as the means ± SD. […]

Pipeline specifications

Software tools SOAPaligner, Primer Express
Applications RNA-seq analysis, qPCR
Organisms Saccharomyces cerevisiae, Penicillium digitatum, Hanseniaspora uvarum, Setaria viridis, Pisum sativum