Computational protocol: Identification and Characterization of a Novel Monoterpene Synthase from Soybean Restricted to Neryl Diphosphate Precursor

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Protocol publication

[…] An EST sequence (TC211089) was identified after a BLAST search of the gene indices of The Institute of Genome Research (TIGR) (http://compbio.dfci.harvard.edu/tgi/), using known monoterpene synthases as the query. Based on the sequence information, the gene-specific primer pair nes-f1 (5′-GCTATGTATGTTGCTAAGACTCTTCAGC-3′) and nes-r1 (5′-AATATGCACTACATGTGCTCAGCAC-3′) was designed for PCR amplification using total RNA from G. max leaves as the template. The PCR program was as follows: 3 min at 94°C followed by 30 cycles of 30 s at 94°C, 50 s at 58°C, and 60 s at 72°C, then 10 min at 72°C. The PCR products were separated by electrophoresis in a 1.0% (w/v) agarose gel and visualized using a JS-380A automatic gel imaging analyzer (PeiQing, Shanghai, P. R. China). The amplified fragments were then subcloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. An internal 1106 bp DNA fragment amplified by the nes-f1 and nes-r1 primers, which showed homology to plant monoterpene synthases, was obtained.RACE was performed with a SMART™ RACE cDNA Amplification Kit (Clontech, USA) to clone the 3′- and 5′-ends of GmNES cDNA following the manufacturer’s instructions. The 3′-end region was amplified by two nested PCR reactions. In the first-round PCR, the primers nes3-f1 (5′-GCTTGTCCATTCATTCTTCCTC-3′) and UPM (supplied in the kit) were used with 3′-RACE-ready cDNA as a template. In the second-round PCR, the primers nes3-f2 (5′-GGACTTGATGGGTCATACATTGA-3′) and NUP (supplied in the kit) were used with the first round products as a template. For 5′-RACE amplification, the two nested primers used were nes5-r1 (5′-TCAGCAAGTTCCTCTAAGCATTC-3′) and nes5-r2 (5′-GAAGAAGTTGGTGGCAGAGG-3′). Based on the sequence information obtained by the 5′- and 3′-RACE reactions, together with the internal sequence, the full-length cDNA was amplified by RT-PCR using the primers nes-f1 (5′-GATGAGGCCAAAAATTGTGC-3′) and nes-r1 (5′-GTGACATCTTTAAGTGCGTGGAC-3′) and then sequenced. The obtained full-length cDNA was designated GmNES.The deduced amino acid sequence was aligned using the Clustal X2.1 program and edited using GeneDOC software (ver 2.6). The phylogenetic tree was created by using the neighbor-joining method and MEGA 4 software. Plastid-targeting peptides and cleavage sites were predicted by the ChloroP tool (ver 1.1). […]

Pipeline specifications

Software tools Clustal W, MEGA, ChloroP
Databases Full Length cDNA
Applications Phylogenetics, Protein sequence analysis
Organisms Glycine max, Nicotiana tabacum, Gossypium hirsutum
Chemicals Terpenes, Salicylic Acid