2.3. Molecular Cloning of Snakehead mc4r and mrap2 Genes

Protein similarity-based blast was performed to search the target genes in the snakehead genome and transcriptome databases [41], using zebrafish mc4r, mrap2a, and mrap2b as references. After searching, we obtained the genomic and transcriptomic sequences of the snakehead mc4r and mrap2 (single copy, showing close relationship with zebrafish mrap2b) genes. Two pairs of primers were designed to validate the two sequences, respectively. Total RNA was isolated from the whole brain with the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol, and 1 μg of the RNA was reverse transcribed to cDNA using Super ScriptTM II RT reverse transcriptase (Takara, Dalian, China). The basic cycling conditions of the PCR were set as follows: A denaturing stage at 94 °C for 30 s, gene-specific annealing temperature for 45 s and an elongation stage at 72 °C for 60 s, a total of 34 cycles. The PCR products were purified from agarose gel using the Universal DNA Purification Kit (TIANGEN, Beijing, China), and then cloned into the pMD-19T vector (TaKaRa). The inserts were sequenced at BGI (Beijing, China), and the primers used to validate mc4r and mrap2 genes were listed in Table S1.