Cloning and Expression Analysis of Body Colour-related Gene mitfa in Rainbow Trout (Oncorhynchus mykiss)
WU Shen-Ji1, HUANG Jin-Qiang1,*, LI Yong-Juan2, ZHANG Qian1, PAN Yu-Cai1, WANG Xiao-Lan1
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Science, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Microphtalmia-associated transcription factor a gene (mitfa) plays an important role in the formation of animal body colour. This study was conducted to explore the regulatory role of mitfa gene in the body colour variation between wild-type (WT) and yellow mutant (YM) rainbow trout (Oncorhynchus mykiss). The full-length sequence of mitfa gene of rainbow trout was obtained by rapid-amplification of cDNA ends (RACE) technique, and the bioinformatics analysis was performed. Furthermore, qRT-PCR was used to investigate the expression levels of mitfa in the different developmental stages and various tissues of WT and YM rainbow trout, respectively. The full-length cDNA of mitfa (GenBank No. MT813117) was 2 700 bp containing 114 bp 5'UTR, 1 374 bp 3'UTR, and 1 212 bp ORF encoding 403 amino acids. Sequence analysis showed that Mitfa protein sequence was more conservative in fishes than that in other vertebrates and the basic helix-loop-helix-leucine zipper (bHLHZip) structural domain was highly conserved among all vertebrates. These results were further confirmed by phylogenetic analysis. qRT-PCR analysis revealed that mitfa expressed at different levels during embryo and post hatch stages, and higher expression levels were detected at the fertilized-stage, 4-cell, 16-cell and multi-cell, which were significantly higher than that of other stages (P<0.05). Moreover, there were extremely significant differences (P<0.01) in the expression of mitfa gene at the same stages between WT and YM rainbow trout, e.g., at the fertilized-stage, 16-cell, multi-cell, blastula, somites, heartbeating, 3 dph (days post hatch), 5 dph, 7 dph, 10 dph, 1 M (month post hatch) and 12 M dorsal skin. mitfa gene expressed in all kinds of tissues of 12 M WT and YM rainbow trout, which expressed the highest amount in dorsal skin (P<0.05), it also highly expressed in ventral skin and muscle, dorasl muscle and eye, but relatively lower in other tissues. The above results indicated that the mitfa gene might be involved in the formation of pigment cells in rainbow trout and closely related to the body colour variation. The results of this study provide basic data for the molecular regulation mechanism of body colour variation and the genetic improvement of body colour in rainbow trout.
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