Computational protocol: A New Set of ESTs from Chickpea (Cicer arietinum L.) Embryo Reveals Two Novel F-Box Genes, CarF-box_PP2 and CarF-box_LysM, with Potential Roles in Seed Development

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Protocol publication

[…] The obtained full length sequences were analyzed using bioinformatic tools at the websites http://www.expasy.org/, http://www.ncbi.nlm.nih.gov/ and Predictprotein (http://www.predictprotein.org/). Conserved domain analysis was performed using Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/). Homologous sequences were searched using BLASTX with default parameters at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for phylogenetic analysis. Multiple sequence alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Phylogenetic relationship tree was constructed using MEGA (ver. 5) [] by Neighbour-Joining (NJ) method utilizing the p-distance method and complete deletion options. The reliability of the obtained tree was tested using a bootstrapping method with 1000 replicates. The homologs for CarF-box_PP2 are as follows: M. truncatula (XP_003590890.1), Glycine max (XP_006588465.1), Phaseolus vulgaris (XP_007143948.1), V. vinifera (CBI38318.3), P. trichocarpa (XP_002324348.1), A. thaliana (NP_973399.1), Zea mays (NP_001150987.1), Oryza sativa (EAY87984.1), Hordeum vulgare (BAJ93930.1) and for CarF-box_LysM, M. truncatula [XP_003591114.1], G. max [NP_001240110.1], P. vulgaris [XP_007144727.1], V. vinifera [XP_002284540.1], A. thaliana [NP_564673.1], H. vulgare [BAJ88125.1], Zea mays [DAA50836.1], O. sativa [NP_001050989.1], Triticum aestivum [ADP02186.1]. [...] Homology modelling for CarF-box_LysM and CarF-box_PP2 was performed using Modeller 9.10 []. Templates used for CarF-box_LysM were 2DJPA (Chain A, The Solution Structure of the LysM domain of Human Hypothetical protein Sb145), 4I6JB (Chain B, A Ubiquitin Ligase-substrate Complex) and 1FQVA (Chain A, Insights into SCF Ubiquitin Ligases from the structure of the Skp1-Skp2 Complex) which showed 38%, 44% and 28% identity, respectively with CarF-box_LysM. For CarF-box_PP2 templates used were 3hq2A, 1p9eA and 2odoA showing 29%, 29% and 28% sequence identity with CarF-box_PP2, respectively. The best models were selected on the basis of the DOPE scores and were refined and energy minimization was done using KoBaMIN [] and quality of the models assessed by PROCHECK [], to obtain a final model. Structures were visualized using PyMOL (http://www.pymol.org/). […]

Pipeline specifications

Software tools PP, BLASTX, Clustal Omega, MODELLER, KoBaMIN, PROCHECK, PyMOL
Databases Pfam ExPASy
Applications Phylogenetics, Protein structure analysis
Organisms Cicer arietinum