Computational protocol: Characterization of the CCT family and analysis of gene expression in Aegilops tauschii

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Protocol publication

[…] The genome and protein sequences of A. tauschii were downloaded from the AGDB database (http://dd.agrinome./org), and a local protein database was established. The hidden Markov model file of the CCT family (PF06203) was downloaded from the Pfam database (http://pfam.sanger.ac.uk/), and the local protein sequence database was retrieved from HMMER software, and redundant sequences were removed. The obtained sequences were confirmed based on the presence of conservative domain of CCT proteins using the SMART database with an E-value cutoff of 1.0 (http://smart.embl-hei-delberg.de).The sequence fragment location of A. tauschii and wheat EST markers information were sourced from the graingene 2.0 database (http://wheat.pw.usda.gov/). The gene structure was analyzed with GSDS software (http://gsds.cbi.pku.edu.cn/). The CCT gene sequences were submitted to the graingene 2.0 database, retrieving the A. tauschii sequences with a similarity of > 95% and E values of 0.0; the related EST sequences were obtained via genome sequence alignment. The CCT genes were named according to their chromosome location, and members of AetCCT were integrated into the related molecular map according to the location information of the sequence fragment and the information of the EST marker. [...] The relative molecular weights, isoelectric points and other information of the CCT protein sequences were obtained from the Editseq software of DNAstar. The conserved motifs among CCT members were identified using the MEME tool (http://meme.nbcr.net/). The parameters were set as follows: width of each motif was 10–300 amino acid residues, maximum number of motifs was 4, and other parameters with default values.The sequences of Arabidopsis, rice and wheat CCT proteins were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/). A total of 98 CCT proteins of related plants were selected for the phylogenetic construction, including 26 from A. tauschii, 22 from Urartu, 30 from rice and 23 from Arabidopsis. Multiple sequence alignments of CCT protein sequences were performed using ClustalX software, and a phylogenetic tree was constructed using the Neighbor-joining Method. The phylogenetic analysis were analyzed using MEGA software with the bootstrap value set to 1,000. The evolution rates (branch models) of each group in the phylogenetic tree were assessed under the tree branch model using PAML software. The positive selection sites were detected with the Branch-site Model. […]

Pipeline specifications

Software tools HMMER, GSDS, MEME, Clustal W, MEGA, PAML
Databases Pfam
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Aegilops tauschii, Triticum aestivum