Localization and Molecular Cloning of the Gene for Melatonin Synthesis in Pigs.

Melatonin is synthesized in multiple tissues and organs of pigs, and existing studies have shown the presence of the melatonin-synthesizing enzyme ASMT protein. However, the genomic information for the gene has been lacking. The aim of this study was to locate the genomic information of the gene in pigs using comparative genomics analysis and then obtain the coding region information through molecular cloning. First, using the NCBI Genome Data Viewer, we found that in most animals, the gene is often located next to the gene, with both genes arranged in the same direction. Similarly, the gene is commonly adjacent to the gene, also in the same orientation. We also discovered that the gene is frequently adjacent to the ASMT gene and arranged in the opposite direction. Using the "three-point localization" principle, we inferred the position of the gene based on the coordinates of and in pigs. Our results revealed that on the pig X chromosome, a gene called LOC110258194 is located next to the and genes, and its arrangement aligns with the gene in other species. Additionally, Ensembl contains a gene, ENSSSCG00000032659, at the same position, with completely overlapping exons, though it is not annotated as . Further analysis using the TreeFam tool from EMBL-EBI and the CDD tool from NCBI revealed that LOC110258194 and ENSSSCG00000032659 do not contain the typical Maf domain of and, thus, should not be annotated as , but rather as the gene. Using a slow-down PCR method for high-GC content genes, we successfully cloned the full CDS region of the pig gene and identified a new transcript missing Exon 6 and Exon 7. This transcript was submitted to NCBI and assigned the GenBank accession number MW847601. Our results represent the first successful localization of the gene in pigs, the first cloning of the gene's coding region, and the first discovery of a new transcript of the pig gene.