Computational protocol: Analyzing the Expression Profile of AREB/ABF and DREB/CBF Genes under Drought and Salinity Stresses in Grape (Vitis vinifera L.)

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Protocol publication

[…] In order to identify the V. vinifera AREB/ABF genes, protein sequences of A. thaliana AREB/ABF genes were used for a BLASTP search against the V. vinifera genome. The bZIP domain was checked in all the grape candidates. All known AREB/ABF gene sequences in the NCBI (http://ncbi.nlm.nih.gov) database along with A. thaliana genes and grape candidates were clustered (), and based on their classification in the cluster, four grape AREB/ABF candidate genes were selected. DREB/CBF genes were identified from V. vinifera genome based on their annotation against the NCBI database. Primers were designed for each of the genes using Primer 3 (http://bioinfo.ut.ee/primer3) and NCBI primer blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast). For family gene distinction, one of the primer pairs was designed from the bZIP domain and the other one was designed from the conserved sequences of the particular gene. Details of the primers are provided in . Quantitative real time PCR (qRT-PCR) reactions were performed using SYBR Green kit (Bio-rad, Hercules, CA). Reactions were performed in a 20 μl volume containing 3 μl of diluted cDNA (5 times diluted from the first strand cDNA stock) and 10 μl 2× iQ SYBR Green Master Mix Supermix as described earlier []. Elongation factor (VvEF1α; accession no. LOC100261589) was used as the internal reference gene (). […]

Pipeline specifications

Software tools BLASTP, Primer3, Primer-BLAST
Application qPCR
Organisms Vitis vinifera
Chemicals Sodium Chloride