Cloning of the pmela Gene in Rainbow Trout (Oncorhynchus mykiss) and Its Expression Analysis in Different Developmental Stages and Tissues

Abstract

Premelanosome protein (pmela) is a key gene in the melanin synthesis pathway, which has an important impact on the skin color of animals. This study was conducted to explore the role of pmela in rainbow trout (Oncorhynchus mykiss) skin color variation. First, the full-length cDNA sequence of pmela was obtained using the rapid amplification of cDNA ends (RACE) technique, and bioinformatic analysis of the encoded protein was performed. Second, quantitative real-time PCR (qRT-PCR) was performed to detect the relative expression of pmela in different developmental stages [from the fertilized-stage to 3 months post-hatching (3 M)] and tissues of wild-type rainbow trout (WT), yellow mutant rainbow trout (YM), and their F1 generation. The results showed that the total length of pmela was 3 476 bp, including a 2 532 bp open reading frame encoding 843 amino acids. Bioinformatic analysis revealed that Pmela was a hydrophobic protein and had a PKD functional domain. Homology alignment showed that the similarity of the Pmela amino acid sequences of rainbow trout and sockeye salmon (Oncorhynchus nerka) was 97.75%. Phylogenetic analysis indicated that rainbow trout was most closely related to sockeye salmon and most distantly to humans (Homo sapiens) and mice (Mus musculus). qRT-PCR results showed that pmela was expressed in all developmental stages of WT and YM, and the expression level of the gene from the fertilized-stage to the 16-cell stage was higher in WT than in YM, whereas the opposite expression pattern was identified after hatching. Moreover, there were significant differences in the expression of pmela at the same stages between WT and YM, including at the fertilized-stage, 4-cell, 16-cell, gastrula, neurula, somites, heartbeating, 1 day post-hatching (dph), 3 dph, 5 dph, 7 dph, 10 dph, 1 M and 2 M. In various tissues, pmela expression was significantly higher in the dorsal skin and eyes of WT and YM than in other tissues. In the hybrid F1 generation, the expression pattern of pmela was similar to those of both parents, and there was a significant difference in expression at the same stages between the F1 generation and the parents in most of the same stages and tissues. The above results indicate that the expression level of pmela is correlated with the skin color of rainbow trout and may be involved in the formation of its skin color, which provides basic data for further studying the role of pmela in the variation of rainbow trout skin color.

Keywords

• rainbow trout
• skin color
• pmela gene
• cloning
• expression analysis