Computational protocol: Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes

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Protocol publication

[…] All of the putative HD-Zip proteins were retrieved from C. morifolium transcriptome data []. Arabidopsis HD-Zip protein sequences were downloaded from The Arabidopsis Information Resource (TAIR) database. The chrysanthemum transcriptome was searched to identify HD-Zip proteins using Basic Local Alignment Search Tool algorithms (tBLASTx) and the published Arabidopsis HD-Zip protein sequences as query sequences. All of the obtained protein sequences were examined for the presence of the HD and LZ domains using the Pfam (http://pfam.sanger.ac.uk/search) and SMART (http://smart.embl-heidelberg.de/) tools. Multiple alignments among the identified CmHB sequences were also performed to avoid repetition. Furthermore, the full open reading frames of the CmHB sequences were obtained via RACE PCR. First-strand cDNA was synthesized using the dT adaptor primer dT-AP and then subjected to nested PCR using the primer pair CmHBx-3-F1/F2 and the adaptor primer AP (). Finally, 17 pairs of gene-specific primers () were designed to amplify complete open reading frames. The amplicons were purified using AxyPrep DNA Gel Extraction Kits (Axygen, Hangzhou, China) and cloned into pMD19-T (TaKaRa, Tokyo, Japan) for sequencing. [...] A phylogenetic tree was constructed with MEGA version 6.0 using the maximum likelihood method []. ClustalW software was employed for multi-sequence alignments of HD-Zip I & II TFs between Arabidopsis and C. morifolium []. Internal branching support was estimated using 500 bootstrap replicates. The theoretical isoelectric point (pI) and molecular weight (Mw) of the CmHB proteins were calculated using the Compute pI/Mw online tool (http://web.expasy.org/compute_pi/), and subcellular localization was predicted with PSORT []. Putative conserved motifs were predicted using the MEME program v4.10.2 [] with the following parameters: any number of repetitions; motif sites, at least 3 sites; optimum motif widths between 6 and 61 residues; and E-value less than 1e-3. All of the motifs identified by MEME were searched in the InterPro database using InterProScan []. The target prediction for miRNA was performed using the psRNATarget online tool []. […]

Pipeline specifications

Software tools BLASTN, TBLASTX, MEGA, Clustal W, PSORT, InterPro, InterProScan, psRNATarget
Databases TAIR Pfam ExPASy
Applications Phylogenetics, Protein sequence analysis, Nucleotide sequence alignment
Organisms Chrysanthemum x morifolium, Arabidopsis thaliana
Diseases Huntington Disease