Molecular Characterization of Guppy Neuropeptide Genes

The open reading frames (ORFs) of guppy it, avt, and itrs were obtained from the genome database (PRJNA238429), and the sequences were confirmed by PCR followed by Sanger sequencing. PCR was performed according to the protocol described in a previous report using cDNA samples from the brain or ovary (38 (link)). Briefly, initial denaturation was performed at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, 55°C to 60°C for 30 s, and 72°C for 1 min. The reaction was terminated with an extension for 5 min at 72°C. The products were purified using a TIANgel Midi purification kit (TIANGEN, Beijing, China), subcloned into the pEASY-T1 cloning vector (TransGen Biotech, Beijing, China) and transformed into DH5α cells. Positive clones containing the inserts of the expected size were selected for sequencing to confirm the results. The confirmed sequences were submitted to the NCBI. All primers used in the present study are listed in Table 1.
The signal peptide and precursor cleavage sites of IT and AVT were predicted by SignalP 5.0 (39 (link)) and NeuroPred software (40 (link)), respectively. Seven putative transmembrane domains were predicted by the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Multiple sequences were aligned and analyzed using Clustal X software (41 (link)), and phylogenetic trees were constructed using MEGA 6 software (42 (link)).