Computational protocol: Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

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Protocol publication

[…] The gene models which were annotated as putative linalool synthase genes (LIN) were obtained from database of VitisNet [] and FLAGdb++ [] both based on grape genome []. The redundant sequences, together with imperfect models caused by insert/deletion mutation and frame shift, were dropped. The amino acid sequences encoded by the rest of putative full length LIN genes were compared with these known plant LINs. Their subcellular localization was predicted through three web tools: ChloroP [], TargetP [] and SignalP []. The gene models that were predicted to contain a chloroplast-located signal peptide were selected as putative grape linalool synthase genes (in short, VvLins). A blast program was performed to screen DFCI Grape Gene Index database [] to find EST fragments corresponding to putative VvLins. The in silico expression profile of VvLins was analyzed.Specific primers covering the open reading frame (ORF) of VvLin genes were designed with the help of Prime 5.0 software package (Premier Biosoft International, Palo Alto, CA, USA) and were then used to amplify their nucleic acid fragments from grape berry cDNA through polymerase chain reaction (PCR).The PCR system of a total volume of 20 μL was composed of 1 μL cDNA template, 1 μL of each primer (20 μM), 10 μL 2× Taq PCR plus master mix and 7 μL ddH2O (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China) according to the protocol of the supplier with slight modification. The temperature gradient PCR was conducted under various conditions: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, primer annealing at 55 ± 10 °C for 1 min and extension at 72 °C for 1 min 30 s at 35 cycles, a final extension at 72 °C for 10 min, and then cooled to 10 °C in a thermal cycler (Techne TC-512 PCR System, Fisher Scientific UK Ltd., Leicestershire, UK). A band of about 1800 bp was gel-purified, subcloned into a pMD19-T Easy vector (Takara, Shiga, Japan) and sequenced using ABI3700 DNA sequencer (Applied Biosystems, Foster City, CA, USA). […]

Pipeline specifications

Software tools ChloroP, TargetP, SignalP
Databases FLAGdb++ VitisNet
Application Protein sequence analysis
Organisms Vitis vinifera
Chemicals Monoterpenes