Computational protocol: Identification of a Novel (-)-5-Epieremophilene Synthase from Salvia miltiorrhiza via Transcriptome Mining

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Protocol publication

[…] Total RNAs from the plant tissues were extracted using CTAB solution (2% CTAB, 2% PVP, 100 mM Tris-HCl, 25 mM EDTA, 2M NaCl, 2% β-mercaptoethanol) and treated with DNase I (Takara) according to the manufacturer’s protocol. Total RNA of 1 μg was reverse-transcribed using the ReverTra Ace qPCR RT Kit (TOYOBO, FSQ-101). Rapid amplification of cDNA end (RACE) was performed by the 5′- and the 3′-Full RACE Core Set Ver.2.0 (Takara) according to manufacturer’s instructions. PCR products were cloned into the pMD18-T Vector (Takara) for sequencing. Full-length cDNAs were obtained by assembling fragments obtained by RACE in combination with annotated unigene sequences.For gene expression analysis, real-time PCR was performed with gene-specific primer pairs on the reverse transcripts derived from total RNAs, using the SYBR® Premix Ex TaqTM II (Perfect Real Time) kit (Takara) on a Mastercycler system (Eppendorf, Germany). ACTIN (HM231319) was used as internal reference (). The relative expression value was calculated via the 2-ΔΔCt method. All PCR primers used in this investigation are listed in Supplementary Table .Terpene synthase sequences from other plant species were obtained through searching the GenBank database at NCBI. Alignment of protein sequences was performed with CLUSTAL W program of MEGA 6 software with default parameters. Phylogenetic tree was built with the neighbor-joining method with MEGA 6 program. Chloroplast signal peptide prediction was performed by SignalP software. […]

Pipeline specifications

Software tools Clustal W, SignalP
Application Phylogenetics
Organisms Salvia miltiorrhiza
Diseases Coronary Disease
Chemicals Diterpenes, Sesquiterpenes, Terpenes, Monoterpenes