Computational protocol: The WRKY transcription factor HpWRKY44 regulates CytP450-like1 expression in red pitaya fruit (Hylocereus polyrhizus)

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Protocol publication

[…] Total RNA was extracted from pitaya pulp samples using the Quick RNA Isolation Kit (Huayueyang, Beijing, China) following manufacturer’s instructions. RNA quality was assessed by gel electrophoresis and spectrophotometry. The extracted RNA was used as template to synthesis cDNAs using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, shiga, Japan).On the basis of the generation of a previous RNA-seq database, a WRKY TF was found to be significantly up-regulated during pulp coloration. As this WRKY displays high degree of sequence homology to Arabidopsis thaliana WRKY44, thus it was termed as HpWRKY44. HpWRKY44 was cloned (primers are listed in ), sequenced and the resulting sequence was queried against the NCBI database in order to identify homologous genes. The theoretical isoelectric point (pI) and mass value for HpWRKY44 protein were determined following the method described at http://web.expasy.org/compute_pi/. Sequences were aligned using CLUSTALW (version 1.83) and GeneDoc software. A phylogenetic tree was constructed using the Neighbor–Joining method in MEGA5.0. […]

Pipeline specifications

Software tools Clustal W, MEGA
Databases ExPASy
Applications Phylogenetics, RNA-seq analysis