Computational protocol: Genome-Wide Identification and Characterization of RBR Ubiquitin Ligase Genes in Soybean

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Protocol publication

[…] To identify the RBR genes in soybean, BLASTP was performed using the putative Arabidopsis and Oryza sativa RBR sequences as seeds to search in soybean genome sequence database (http://www.phytozome.net/soybean). Protein sequences were evaluated using Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/), and the sequences having no RBR domains or containing less than 180 residues were discarded.The sequences were aligned using CLUSTALX2.1 and the alignments were edited with the GeneDoc 2.7 sequence editor. Maximum Likelihood fits of 48 different amino acid substitution models were tested using MEGA5 , and models with the lowest BIC (Bayesian Information Criterion) scores are considered to best describe the substitution pattern and the parameters were used for the phylogenetic tree construction. Maximum-likelihood (ML) trees were obtained using PhyML (approximate likelihood ratios analysis) . The neighbor-joining (NJ) trees were constructed in MEGA5. All phylogenetic analyses had been done with above two methods of ML and NJ with 1000 bootstraps. The dendrograms were edited with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).The duplication patterns of the soybean RBR genes were analyzed based on their gene locations in the soybean genome. Tandem duplicated genes are located next to each other, and block duplications were detected with Synteny plot in Plaza (http://bioinformatics.psb.ugent.be/plaza/synteny). First, the RBR genes were analyzed with BLAST in Plaza, and then the gene or gene family IDs were putted into Synteny plot to detect the duplication patterns.The protein domains and conserved motif were analyzed using pfam (http://pfam.sanger.ac.uk/), SMART (http://smart.embl-heidelberg.de/) and MEME (http://meme.nbcr.net/meme/). Coiled-coil domains in these RBR domain-containing protein sequences were predicted with MARCOIL , and COILS . […]

Pipeline specifications

Software tools BLASTP, Clustal W, MEGA, PhyML, FigTree, MARCOIL
Databases Pfam Phytozome
Applications Phylogenetics, Protein structure analysis
Organisms Glycine max